Methods and compositions for impairing multiplication of HIV-1

ABSTRACT

A composition which elicits antibodies to greater than 95%, and even greater than 99%, of the known variants of HIV-1 Tat protein contains at least one peptide or polypeptide of the formula of Epitope I (based on amino acids 2-10 of HIV-1 Tat consensus sequence) and optionally one or more of a peptide or polypeptide of Epitope II (based on amino acids 41 to 51 of that sequence), of Epitope III (based on amino acids 52-62 of that sequence), or of Epitope IV (based on amino acids 62 through 72 of that sequence with a C-terminal Pro). Vaccinal and pharmaceutical compositions can contain the antibodies induced by the peptide compositions for use in passive therapy. Diagnostic compositions and uses are described for assessing the immune status of vaccinated patients

CROSS-REFERENCE TO OTHER INVENTIONS

[0001] This is a divisional of U.S. patent application Ser. No.09/451,067, filed Nov. 30, 1999, which is a divisional of U.S. patentapplication Ser. No. 09/113,921, filed Jul. 10, 1998, now U.S. Pat. No.6,193,981, issued Feb. 27, 2001, which is a continuation-in-part of U.S.patent application Ser. No. 08/893,853, filed Jul. 11, 1997, now U.S.Pat. No. 5,891,994, issued Apr. 6, 1999

BACKGROUND OF THE INVENTION

[0002] The present invention relates generally to compositions andmethods useful for inhibiting the multiplication of humanimmunodeficiency virus-1 (HIV-1) in infected patients, symptomatic orasymptomatic, and for attenuating HIV-1 multiplication during primaryinfection in previously uninfected subjects, thus minimizing progressionto AIDS.

[0003] High plasma levels of human immunodeficiency virus type 1 (HIV-1)RNA are found during primary infection with HIV-1, the seroconversionillness, (C. Baumberger et al, AIDS, 7:(suppl 2):S59 (1993); M. S. Saaget al, Nature Med., 2:625 (1996)), after which they subside as theimmune response controls the infection to a variable extent. Postseroconversion, lower but detectable levels of plasma HIV-1 RNA arepresent, and these levels rise with disease progression to again attainhigh levels at the AIDS stage (M. S Saag et al, Nature Med., 2:265(1996)). Approximately 50% of subjects have a symptomatic illness atseroconversion (B. Tindall and D. A. Cooper, AIDS, 5:1 (1991)) andsymptomatic seroconversion is associated with an increased risk for thedevelopment of AIDS, probably because a severe primary illness is likelyrelated to an early and extensive spread of HIV.

[0004] Inhibition of viral multiplication during the initial infectionwill likely reduce the subsequent development of chronic viremia leadingto AIDS. Current medical practice, with administration of antiviraldrugs for defined “at risk” situations, such as needle sticks withcontaminated blood or pregnancy in HIV infected mothers, supports thisconcept.

[0005] Post seroconversion levels of HIV-1 RNA in plasma have proven tobe the most powerful prognosticator of the likelihood of progression toAIDS (J. W. Mellors et al, Science, 272:1167 (1996); M. S. Saag et al,Nature Med., 2:265 (1996); R. W. Coombs et al, J. Inf. Dis., 174:704(1996); S. L. Welles et al, J. Inf. Dis., 174:696 (1990)). Othermeasures of viral load, such as cellular RNA (K. Saksela et al, ProcNatl Acad. Sci. USA, 91:1104 (1994)) and cellular HIV proviral DNA (T-H. Lee et al, J Acq. Imm. Def. Syndromes, 7:381 (1994)) similarlyestablish the importance of the initial infection in establishing viralloads that determine future disease progression.

[0006] Thus, any intervention that inhibits HIV-1 infectivity duringinitial infection and/or lowers viral load post sero-conversion islikely to have a favorable influence on the eventual outcome, delayingor preventing progression to AIDS.

[0007] A variety of methods are now employed to treat patients infectedwith human immunodeficiency virus (HIV-1), including treatment withcertain combinations of protease inhibitor drugs. Unfortunately,however, this type of treatment is associated with serious side effectsin some patients. Alternatively, vaccines are under development forcontrol of the spread of HIV-1 to uninfected humans. However, thiseffort has largely been directed to proteins of the virus, expressed onthe surface of infected cells, which are recognized by cytotoxic T cellswith elimination of the infected cells, while free virus is blocked andcleared by antibody to surface antigens of the virion. Limitations ofthis mode of vaccination are readily apparent for HIV-1, which hasdemonstrated a great diversity in immunogenic viral epitopes and rapidmutational variations that occur within and between individuals (B. D.Preston et al., Science, 242:1168(1988); J. D. Roberts et al., Science,242:1171 (1988); A. R. Meyerhans et al., Cell, 58:901 (1989); K. Kusumiet al., J. Virol., 66:875 (1992); B. A. Larder et al., Science, 243:1731(1989); M. S. Sang et al., N. Engl. J. Med., 329:1065 (1993); M. A.Sande, et al., JAMA, 270:2583 (1993), M. Seligmann et al., Lancet,343:871 (1994); G. Meyers et al., Human retroviruses and AIDS 1993, I-V.A compilation and analysis of nucleic acid and amino acid sequences. LosAlamos National Laboratory, Los Alamos, N. Mex.)

[0008] Variation in strains of HIV-1 and frequent mutations of virionproteins have prevented successful application of conventional vaccineapproaches (W. E. Paul, Cell, 82:177 (1995); J. E. Osborn, J. Acq. Imm.Def. Syndr. Hum. Retrovirol., 9:26 (1995)). Mutation and selection ofresistant variants is the central problem in developing a successfulHIV-1 vaccine (M. D. Daniel et al., Science, 258:1938 (1992); N. L.Letvin, N. Engl. J. Med., 329:1400 (1993); M. Clerici et al., AIDS,8:1391 (1994); S. M. Wolinsky et al, Science, 272:537 (1996)).

[0009] Other approaches to HIV-1 treatment have focused on thetransactivating (tat) gene of HIV-1, which produces a protein (Tat)essential for transcription of the virus. The tat gene and its proteinhave been sequenced and examined for involvement in proposed treatmentsof HIV (see, e.g., U.S. Pat. No. 5,158,877; U.S. Pat. No. 5,238,882;U.S. Pat. No. 5,110,802; International Patent Publication No.WO92/07871, published May 14, 1992; International Patent Publication No.WO91/10453, published Jul. 25, 1991; International Patent PublicationNo. WO91/09958, published Jul. 11, 1991; International PatentPublication No. WO87/02989, published May 21, 1987). Tat protein isreleased extracellularly, making it available to be taken up by otherinfected cells to enhance transcription of HIV-1 in the cells and to betaken up by noninfected cells, altering host cell gene activations andrendering the cells susceptible to infection by the virus. Uptake of Tatby cells is very strong, and has been reported as mediated by a shortbasic sequence of the protein (S. Fawell et al., Proc. Natl. Acad. Sci.,USA, 91:664-668 (1994)).

[0010] International Patent Publication No. WO92/14755, published Sep.3, 1992, relates to the Tat protein and to the integrin cell surfacereceptor capable of binding to the Tat protein. Two Tat sequences thatbind integrin are identified, which are the basic region or domain whichis the dominant binding site for the integrin, having a peptide sequenceof -Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg- (SEQ ID NO: 4), as well as-Gly-Arg-Gly-Asp-Ser-Pro- (SEQ ID NO: 5). This specificationdemonstrates that a number of peptides corresponding to these Tatsequences and the corresponding integrins block in vitro cell binding toTat coated plates, as do antibodies to the appropriate integrins.However, the specification also shows that these reagents do not blockuptake of functional Tat by cells (see Example 9 in WO92/14755), thusnullifying the proposed mechanism of action for therapeutic benefit inHIV infection. The Tat sequences described in this internationalapplication are distinct from the peptide immunogens of the presentinvention.

[0011] Both monoclonal and polyclonal antibodies to Tat protein havebeen readily produced in animals and shown to block uptake of Tatprotein in vitro (see, e.g., D. Brake et al, J. Virol., 64:962 (1990);D. Mann et al, EMBO J., 10:1733 (1991); J. Abraham et al, cited above;P. Auron et al, cited above; M Jaye et al, cited above; G. Zauli et al,cited above). More recent reports showed that monoclonal or polyclonalantibodies to Tat protein added to tissue culture medium attenuatedHIV-1 infection in vitro (L. Steinaa et al, Arch. Virol., 139:263(1994); M. Re et al, J. Acq. Imm. Def. Syndr. Hum. Retrovirol., 10:408(1995); and G. Zauli et al, J. Acq. Imm. Def. Syndr. Hum. Retrovirol.,10:306 (1995)).

[0012] The inventor's own publication (G. Goldstein, Nature Med., 2:960(1996); see also, International Patent Publication No. WO95/31999,published Nov. 30, 1995) reviewed the evidence indicating that secretionof HIV-1 Tat protein from infected cells and uptake by both infected anduninfected cells was important for the infectivity of HIV-1. Previousstudies also showed that antibodies to Tat protein in vitro blockeduptake of Tat and inhibited in vitro infectivity. Goldstein proposedactive immunization of mammals to induce antibodies to HIV-1 Tat proteinas a potential AIDS vaccine.

[0013] Despite the growing knowledge about HIV-1 disease progression,there remains a need in the art for the development of compositions andmethods for treatment of HIV-1, both prophylactically andtherapeutically, which are useful to lower the viral levels of HIV-1 forthe treatment and possible prevention of the subsequent, generallyfatal, AIDS disease

SUMMARY OF THE INVENTION

[0014] In one aspect, the invention provides as a novel compositioncomprising a peptide or polypeptide, which comprises an amino acidsequence selected from the formula referred to as Epitope I:R1-Val-Asp-Pro-Y-Leu-Glu-Pro-R2 (SEQ ID NO: 36), wherein Y is variouslyArg, Lys, Ser or Asn. The N-terminal R1 may represent hydrogen (i.e.,the hydrogen on the unmodified N terminal amino acid), or a lower alkyl,or a lower alkanoyl. R1 may also include a sequence of between 1 toabout 5 amino acids, optionally substituted with a lower alkyl or loweralkanoyl. In one embodiment, R1 is -X-Pro-, wherein X is Glu or Asp.Preferably, R1 represents 2 amino acids. The C-terminal R2 can alsorepresent the hydroxyl group on the C terminal amino acid or an amide.To enhance titer R2 is preferably a sequence of between 1 to about 14additional amino acids amidated at the carboxyl terminus. In a preferredembodiment, R2 is -Trp-Lys-His-Pro-Gly-Ser- amide (SEQ ID NO: 10). Thepeptides or polypeptides of these compositions are producedsynthetically or recombinantly. This composition may take the form ofone or more of the above-described peptides expressed as a syntheticpeptide coupled to a carrier, or expressed as a multiple antigenicpeptide, or the selected peptides may be expressed within arecombinantly produced protein. This composition is designed to induceantibodies reactive with greater than 95% of the known variants of theHIV-1 Tat protein.

[0015] In another aspect, the above-described composition furthercontains one or more additional peptide or polypeptide(s) whichrepresent other amino acid sequences which correspond to amino acidresidues 2 or 4 to 10 of an HIV-1 Tat protein. These optional amino acidsequences are described in detail below. These sequences are preferablyfrom an HIV-1 strain with a Tat protein variant at that location.

[0016] In another aspect, the invention provides a novel compositioncomprising a peptide or polypeptide of the formula referred to asEpitope II: R3-Lys-X-Leu-Gly-Ile-Ser-Tyr-Gly Arg-Lys-Lys-R4 (SEQ ID NO:37). According to this formula, X is Gly or Ala. The N terminal R3 mayrepresent hydrogen (i.e., the hydrogen on the unmodified N terminalamino acid), or may be a lower alkyl, or a lower alkanoyl. R3 may alsoinclude a sequence of between 1 to about 5 amino acids, optionallysubstituted with a lower alkyl or lower alkanoyl. The C terminal R4 maybe the free hydroxyl of the C terminal amino acid, or an amide, or asequence of one or up to about 5 additional amino acids, optionallysubstituted with an amide. The peptides or polypeptides of thesecompositions are produced synthetically or recombinantly, provided thatthe recombinant Epitope II peptide is situated at the C terminus of therecombinant protein. This composition may take the form of one or moreof the above-described peptides expressed as a synthetic peptide coupledto a carrier, or expressed as a multiple antigenic peptide. Thiscomposition is designed to induce antibodies reactive with greater thanabout 95% of the known variants of HIV-1 Tat protein.

[0017] In yet a further aspect, this invention provides a compositioncomprising a peptide or polypeptide of the formula referred to asEpitope III: R5-Arg-Arg-X-Z-A-Y-Ser-R6 (SEQ ID NO: 38), wherein X isselected from the group consisting of Ala, Pro, Ser and Gln; wherein Yis selected from the group consisting of Asp, Asn, Gly and Ser; whereinZ is selected from the group consisting of Pro and His; and wherein A isselected from the group consisting of Gln and Pro. The N terminal R5 ishydrogen, a lower alkyl, a lower alkanoyl, or a sequence of between 1 toabout 3 amino acids, optionally substituted with a lower alkyl or loweralkanoyl. In a preferred embodiment R5 is -Gln-Arg-, optionally modifiedas above. The C terminal R6 is either a free hydroxyl or an amide. Apreferred embodiment of such a composition contains at least threeEpitope III peptides, i.e., -Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- (aminoacids 54-62 of SEQ ID NO: 1), -Gln-Arg-Arg-Arg-Ala-His-Gln-Asp-Ser-(amino acids 2-10 of SEQ ID NO: 65), and-Gln-Arg-Arg-Arg-Ala-Pro-Pro-Asp-Ser- (amino acids 264-272 of SEQ ID NO:3), optionally modified as above. Other peptides or polypeptidesrepresentative of amino acids 56-62 of Tat, but having differentsequences from that of the above formula may also be included in thecomposition. The peptides or polypeptides of these compositions areproduced synthetically or recombinantly. This composition may take theform of one or more of the above-described peptides expressed as asynthetic peptide coupled to a carrier, or expressed as a multipleantigenic peptide, or the selected peptides may be expressed within arecombinantly produced protein. This composition is designed to induceantibodies reactive with greater than about 75% of all known variants ofHIV-1 Tat protein.

[0018] In still a further aspect, this invention provides a compositioncomprising a peptide or polypeptide of the formula referred to asEpitope IV: R7-Ser-Gln-X-His-Gln-Y-Ser-Leu-Ser-Lys-Gln-Pro-R8 (SEQ IDNO: 39), wherein X is selected from the group consisting of Asn and Thr;and wherein Y is selected from the group consisting of Ala and Val. TheN terminal R7 may be hydrogen, a lower alkyl, a lower alkanoyl, or asequence of between 1 to about 3 amino acids, optionally substitutedwith a lower alkyl or lower alkanoyl. The C terminal R8 may be a freehydroxyl, an amide, or a sequence of one or up to about 3 additionalamino acids, optionally substituted with an amide. A preferred EpitopeIV peptide is -Ser-Gln-Thr-His-Gln-Ala-Ser-Leu-Ser-Lys-Gln-Pro- (SEQ IDNO: 40). The peptides or polypeptides of these compositions are producedsynthetically or recombinantly. This composition may take the form ofone or more of the above-described peptides expressed as a syntheticpeptide coupled to a carrier, or expressed as a multiple antigenicpeptide, or the selected peptides may be expressed within arecombinantly produced protein. This composition is designed to induceantibodies reactive with greater than 64% of all known variants of HIV-1Tat protein.

[0019] In still another aspect, this invention provides compositiondescribed above that contains peptides or polypeptides which compriseone or more Epitope I peptides in combination with one or more EpitopeII peptides, and/or one or more Epitope III peptides, and/or one or moreEpitope IV peptides. Such compositions can combine appropriate Epitopepeptides, so as to provide for a composition than induces antibodiesreactive with greater than about 99% of all known HIV-1 Tat proteins.

[0020] In yet a further aspect, the invention provides a synthetic genewhich encodes sequentially a peptide or polypeptide that contains atleast one Epitope I amino acid sequence defined above, optionally with acarboxy terminal Epitope II peptide, or contains at least two Epitope Iamino acid sequences. The synthetic gene may contain each amino acidsequence separated by a spacer sequence, or may express eachpeptide/polypeptide in an open reading frame with a carrier protein. Thesynthetic gene may be separated from the carrier protein by a spacer ifthe spacer is fused to an Epitope I sequence, leaving an Epitope IIsequence at the carboxy terminus of the recombinant protein. Furtherembodiments include multiple Epitope I peptides fused together and tothe carrier protein.

[0021] In yet a further aspect, the invention provides a syntheticmolecule, e.g., a vector, comprising the above-described synthetic gene,operatively linked to regulatory nucleic acid sequences, which directand control expression of the product of the synthetic gene in a hostcell.

[0022] In another aspect, the invention provides a recombinant viruswhich contains the above described synthetic gene or synthetic molecule,which virus is capable of expressing multiple copies of the product ofthe gene or molecule in a host cell. The virus is non-pathogenic tohumans.

[0023] In yet another aspect, the invention provides a commensalbacterium which contains the above described synthetic gene or syntheticmolecule, which bacterium is capable of expressing multiple copies ofthe product of the gene or molecule and inducing antibodies in amammalian host.

[0024] In still a further aspect, the invention provides an isolatedantibody composition which is directed against a peptide or polypeptideof the compositions described above. Antibodies may also be obtainedagainst multiple components of the compositions described above. Thisantibody is produced by immunizing a mammal with a peptide/polypeptidecomposition of the invention, a synthetic gene or synthetic molecule ofthe invention; a recombinant virus or commensal bacterium of theinvention; and isolating and purifying antibody from said immunizedmammal. Alternatively, the antibody may be a polyclonal antibody, amonoclonal antibody, a chimeric antibody, a humanized antibody, a humanantibody, or mixtures thereof.

[0025] Thus, another aspect of the invention is a pharmaceuticalcomposition useful for inducing antibodies that react with greater than95%, and preferably greater than 99%, of the known HIV-1 Tat proteins.These induced antibodies can impair the

[0026] In yet a further aspect, the invention provides a method fordetecting the titers and reactivity patterns of antibodies in subjectsvaccinated with the compositions of this invention. The method includesthe steps of incubating dilutions of the subject's biological fluid,e.g. serum, with plates or beads on which are bound one or more peptidesof the Epitopes I through IV, washing away unbound biological materials,and measuring any antibody binding to the peptides with labeled reagent,e.g., an anti-human immunoglobulin to which is associated an enzyme.Depending on the type of label employed, the signal produced by thelabel may be evoked by further adding a substrate which reacts with theenzyme, e.g., producing a color change. Other conventional labels mayalso be incorporated into this assay design.

[0027] Other aspects and advantages of the present invention aredescribed further in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 illustrates an HIV-1 Tat protein consensus sequence (SEQ IDNO: 1), based on Tat protein sequences of 31 known HIV-1 strains foundin the common B subtype (NIH Los Alamos database). The amino acidpositions in which variations appear are in lower case letters.

[0029] FIGS. 2A-2C illustrate a synthetic gene which encodes a fusionprotein (SEQ ID NO: 3) of this invention, described in detail in Example5 below.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention provides a solution to the above-statedproblem by providing compositions which induce antibodies in uninfectedor early stage infected subjects still capable of mounting an immuneresponse to an immunogen, the antibodies reacting with greater than 95%,and preferably, greater than 99% of known HIV-1 Tat protein variants.The induced antibodies can inhibit multiplication of HIV-1. Thisprevents further disease progression to AIDS. Antibody compositions arealso provided for use in infected or non-infected humans, who areincapable of mounting an effective or rapid immune response to HIV-1infection. These compositions are capable of reacting with greater than95%, and preferably greater than 99%, Tat proteins thus reducing virallevels of HIV-1. These antibodies are useful in both therapeutic andprophylactic contexts to control the development of AIDS in a largepopulation exposed to, or infected by, HIV-1 strains which produce uponinfection immunologically distinct Tat proteins.

[0031] The compositions of the present invention, which are based onpeptides provided by certain epitopes of HIV-1 Tat protein, may beproteinaceous in nature, or may be nucleic acid compositions whichencode the peptides and polypeptides that induce antibodies to Tat,which in turn impair multiplication of HIV-1.

[0032] HIV-1 Tat protein is produced from two exons: Exon 1 encodes a 72amino acid (aa) protein (see FIG. 1, SEQ ID NO: 1) which may beexpressed without splicing or be spliced with the approximately 29 aminoacid peptide encoded by Exon 2 to produce an approximately 101 aminoacid peptide. Since the 72 amino acid product of Exon 1 is capable aloneof cellular uptake and activation, it is essential that antibodies reactwith and interdict intercellular transport of the 72 amino acid peptide.HIV-1 Tat contains Cys at aa positions 22 and 37 of Exon 1 (SEQ ID NOS:1 and 2), and 5 additional Cys between these. This region of the peptideis termed the Cys rich region and has Cys-Cys covalent linkagesproducing complex tertiary structure. The scientific literature hasindicated that this region does not appear to be immunogenic.

[0033] The inventor has identified epitopes, i.e., binding regions,recognized by antibodies (antigenic sequences) in the N-terminal linearsequence 1-21 (22 aa) and the C-terminal linear sequence 38-72 (35 aa)of Exon 1 (SEQ ID NO: 2) or other Tat sequence variants. Immunogenicregions of these larger sequences were identified by the inventor andare underlined in the N terminal and C terminal consensus sequences ofExon 1 below: Epitope I was identified as the nine amino acid sequenceof aa positions 2-10 of Exon 1. Epitope II was identified as the elevenamino acid sequence of aa positions 41-51 of Exon 1. Epitope III wasidentified as the 7 amino acid sequence of aa positions 56-62 of Exon 1.Epitope IV was identified as the twelve amino acid sequence of aapositions 62-73 of Tat, including the the first Pro (aa 73) of Exon 2and overlaps Ser 62 of Epitope III (dotted underlining).  1 MetGlu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro LysThr (SEQ ID NO: 54)  21            38          41 Ala...........Phe IleThr Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys (SEQ ID NO: 55) 51                 56                      62 Lys Arg Arg Gln ArgArg Arg Ala Pro Gln Asp Ser Gln Thr His Gln Val Ser Leu                                            -----------------------------------         72 Ser Lys Gln Pro ---------------

[0034] The term “Tat sequence variant” means a polypeptide or peptidecontaining Tat protein amino acid residues, or a sequence from anotherHIV-1 strain Tat protein that is substantially similar to the sequenceof SEQ ID NO: 1. Each variant may differ from the consensus sequence ofFIG. 1 (SEQ ID NO. 1) and/or from another variant by at least one aminoacid change within the residues of interest for Epitopes I through IV.This change may provide the same or different antigenic specificity tothat particular Tat Epitope when added to the composition of theinvention.

[0035] A. Epitope I Immunogenic Compositions

[0036] Therefore, in one embodiment, the present invention provides acomposition containing a non-naturally occurring peptide or polypeptide,which comprises one or more Epitope I amino acid sequences. TheseEpitope I sequences elicit a specific humoral immune response (for thepurpose of this invention) in a mammal exposed to the Epitope Isequences in vivo. The Epitope I amino acid sequences correspond toamino acid residues 2-10 or 4-10 of the Tat consensus sequence (SEQ IDNO: 1) of FIG. 1 which is derived from a number of “Tat sequencevariants”

[0037] Epitope I defines peptides of the general formula:R1-Val-Asp-Pro-Y-Leu-Glu-Pro-R2 (SEQ ID NO: 36). The N-terminal R1 mayrepresent the hydrogen on the unmodified N terminal amino acid Val, orR1 may be a lower alkyl, or a lower alkanoyl attached to the Val. R1 mayalso include a sequence of between 1 to about 5 amino acids, optionallysubstituted with a lower alkyl or lower alkanoyl. In multiplication ofHIV-1. The pharmaceutical composition comprises at least one of therecombinant or synthetic peptide/polypeptide compositions describedabove, the synthetic gene/molecule described above, the recombinantvirus described herein; or the commensal bacterium described herein, ina pharmaceutically acceptable carrier.

[0038] Still a further aspect of the invention is a pharmaceuticalcomposition useful for impairing the multiplication of HIV-1, thiscomposition containing an above described antibody composition.

[0039] In yet a further aspect of the invention, a method for reducingthe viral levels of HIV-1 involves exposing a human to antibody-inducingpharmaceutical compositions described above, actively inducingantibodies that react with most HIV-1 Tat proteins, and impairing themultiplication of the virus in vivo. This method is appropriate for anHIV-1 infected subject with a competent immune system, or an uninfectedor recently infected subject. The method induces antibodies which reactwith HIV-1 Tat proteins, which antibodies reduce viral multiplicationduring any initial acute infection with HIV-1 and minimize chronicviremia which leads to AIDS.

[0040] In still another aspect, the invention provides a method forreducing the viral levels of HIV-1 by administering to a human, who isincapable of mounting an effective or rapid immune response to infectionwith HIV-1, a pharmaceutical composition containing the antibodycompositions described above. The method can involve chronicallyadministering the composition.

[0041] Yet other aspects of the invention include methods for producingthe compositions described above, as well as host cells transfected withsuch compositions.

[0042] Still another aspect of this invention is a kit useful for themeasurement and detection of titers and specificities of antibodiesinduced by vaccination with the compositions described above. The kit ofthe invention includes peptides of Epitopes I through IV, and coatedsolid supports, a labelled reagent for detecting the binding ofantibodies to these peptides, and miscellaneous substrates and apparatusfor evoking or detecting the signals provided by the labels, as well asconventional apparatus for taking blood samples, appropriate vials andother diagnostic assay components. one embodiment, R1 is -X-Pro-,wherein X is Glu or Asp. The C-terminal R2 can represent the hydroxylgroup on the C terminal amino acid Pro, or R2 can be an amide on thePro; alternatively, R2 is a sequence of between 1 to about 14 additionalamino acids, optionally amidated at the carboxyl terminus. The X and Ypositions represent common variants of this Epitope peptide, wherein Xis Glu (90%) or Asp (10%), and Y is variously Arg (74%), Lys (11%), Ser(9%) or Asn (4%). Peptides containing Glu or Asp at position X induceantibodies that effectively cross-react with the other variant.Alternatively, peptides omitting the X-P effectively induce antibodiesto Val-Asp-Pro-Arg-Leu-Glu-Pro (amino acids 4-10 of SEQ ID NO: 1). Adesirable immunogen of the formula Glu-Pro-Val-Asp-Pro-Lys-Leu-Glu-Pro(SEQ ID NO: 56) reacts/cross-reacts with greater than 95% of known HIV-1Tat proteins, while an immunogen of the formulaVal-Asp-Pro-Lys-Leu-Gly-Pro (SEQ ID NO: 57) reacts/cross-reacts withgreater than 97% of known HIV-1 Tat proteins.

[0043] Antibodies to all four position Y variants are generated by usingall four as immunogens. Alternatively, cross-reactivity permits areduction to two or even one immunizing sequence to induce reactivity toall four position Y variants. As discussed in detail in the Examplesbelow, the reactivities of antibodies induced by the EpitopeI-containing peptide: Val-Asp-Pro-Y-Leu-Glu-Pro-Typ-Lys-His-Pro-Gly-Ser-(SEQ ID NO: 58), where Y is Arg, Lys, Ser or Asn, are reported in Table1 below. Dark shading shows self-reactivity, pale shading showssignificant (40%) cross-reactivity. TABLE 1 Immunizing Peptides DetectorPeptides (GMT (% self-binding)) Position Y Arg Lys Ser Asn Arg  77,000(100)  10,000 (13) 10,000 (13)  9,000 (12) Lys* 51,000 (62)  82,000(100) 35,000 (43) 45,000 (55) Ser 8,000 (6)  8,000 (6) 128,000 (100)14,000 (11) Asn* 17,000 (13) 12,000 (9) 61,000 (46) 134,000 (100)

[0044] Preferably a composition of this invention contains one or moreof the following Epitope I peptides or polypeptides:

[0045] R1-Val-Asp-Pro-Arg-Leu-Glu-Pro-R2 (SEQ ID NO: 6);

[0046] R1-Val-Asp-Pro-Lys-Leu-Glu-Pro-R2 (SEQ ID NO: 7);

[0047] R1-Val-Asp-Pro-Ser-Leu-Glu-Pro-R2 (SEQ ID NO: 8);

[0048] R1-Val-Asp-Pro-Asn-Leu-Glu-Pro-R2 (SEQ ID NO: 9).

[0049] As demonstrated above, the immunogen in which Y is Lys (SEQ IDNO: 7) induces antibodies with good reactivity with the three othervariants. No immunogen induced high titer antibodies with goodcross-reactivity with the variant in which Y was Ser. Thus an immunogenof Epitope I in which Y was Lys (SEQ ID NO: 7) may suffice for fullcross-reactivity to all four position Y variants, and may be used alonein an immunogenic composition. While this pattern of response of thepeptide in which Y is Lys occurs in the majority of tests to date, itshould be expected by one of skill in the art, that some differences incross-reactivity from the results above may occur in some test samples

[0050] Alternatively, compositions of this invention comprise two, threeor all four of these amino acid sequences (SEQ ID NOS: 6-9).Alternatively, a combination of Epitope I immunogens in which Y was Lysand in which Y was Asn (SEQ ID NO: 6) should provide somewhat bettertiters for Epitope I variants in which Y was Ser or Y was Asn.

[0051] Still another alternative Epitope I peptide immunogen contains atposition Y an ornithine, since the ornithine side chain is similar tolysine with one less —CH₂—. This Epitope I sequence may provide evenmore cross-reactivity, and may be used alone or in combination withother Epitope I immunogens.

[0052] According to the formula of Epitope I above, the seven amino acidresidues which form the minimum reactive Epitope I sequences, may beflanked by other amino acids, so that the entire Epitope I sequence maybe between 7 and about 25 amino acids in length. As indicated in Example1 below, the identity of the flanking amino acids is not essential tothe biological function of the Epitope I immunogen. In particularadditional amino acids on the N-terminus of Epitope I sequences do notaffect immunogenicity. Thus, the N-terminal R1 of Epitope I may beselected from the group consisting of a free N terminal amino acidhydrogen, a lower alkyl (i.e., C1-C10 alkyl), a lower C1-C10 alkanoyl,such as an acetyl group, or a sequence of between 1 to about 5 aminoacids. Preferably, R1 represents 2 amino acids.

[0053] Additional amino acids on the C-terminus of the Epitope I minimumsequence enhance antibody titer. Epitope I immunogens require at leasttwo amino acid extensions at the C-terminus for optimal immunogenicityand are immunogenic when present within extended amino acid sequence.Thus, while the C-terminal R2 can be a simple free hydroxyl group, itcan also be a C terminal amide. However, to enhance titer, R2 ispreferably a sequence of between 1 to about 14 additional amino acidsamidated at the carboxyl terminus In a preferred embodiment, R2 is-Trp-Lys-His-Pro-Gly-Ser-amide (SEQ ID NO: 10).

[0054] The above-described Epitope I composition of the invention maycontain a number of additional peptides or polypeptides, which containother sequences which correspond to amino acid residues between aa2-aa10 of SEQ ID NO: 1, but are derived from other Tat variants which donot cross-react well with antibodies to the Epitope I compositionsdescribed above. These additional peptides and polypeptides are referredto as “optional Epitope Ia immunogens”. For example, optional Epitope Iaimmunogens which can be present in compositions of this invention, cancontain at least one copy of at least one of the following amino acidsequences (SEQ ID NOS: 11 through 18, respectively):

[0055] R1-Gly-Pro-Arg-Leu-Glu-Pro-R2;

[0056] R1-Ala-Pro-Arg-Leu-Glu-Pro-R2;

[0057] R1-His-Pro-Arg-Leu-Glu-Pro-R2;

[0058] R1-Asp-Pro-Gly-Leu-Glu-Pro-R2;

[0059] R1-Asp-Pro-Arg-Ile-Glu-Pro-R2;

[0060] R1-Asp-Pro-Arg-Leu-Gly-Pro-R2;

[0061] R1-Asp-Pro-Arg-Leu-Glu-Ala-R2; and

[0062] R1-Asn-Pro-Ser-Leu-Glu-Pro-R2.

[0063] The Epitope I compositions of this invention may contain multiplecopies of a single peptide, or multiple copies of different Epitope Ipeptides, including optionally Epitope Ia peptides, in any order, ormultiple copies of at least two of these peptides. In one embodiment, atleast one copy of all four amino acid sequences (SEQ ID NOS: 6-9) arepresent.

[0064] As described in more detail below, the Epitope I and Ia peptidesor polypeptides of these compositions are produced synthetically orrecombinantly. The Epitope I immunogens can be expressed as syntheticpeptides coupled to a carrier protein. The Epitope I immunogens may alsobe expressed as multiple antigenic peptides, optionally coupled to acarrier protein. Alternatively, the Epitope I immunogens may beexpressed within recombinantly produced protein, optionally co-expressedor fused in frame with a carrier protein.

[0065] Epitope I compositions demonstrate a biological activity ofinducing in an immunized, immune competent mammal, i.e., a non-infectedhuman, or an asymptomatic infected human, an active humoral immuneresponse (i.e., antibodies) that is directed against greater than 95%,and preferably greater than 99%, of the known variants of Tat proteinsof HIV-1. The end result of such treatment is an impairment of themultiplication of HIV-1 in an acute infection, thereby preventing highpost-seroconversion plasma levels of HIV-1 that are associated withprogression to AIDS. Active induction of antibodies in the earlyasymptomatic phase of HIV infection may reduce viral multiplication,lower the plasma viral load and reduce the likelihood of progression toAIDS. The composition which contains at least one Epitope I immunogen upto all four of the SEQ ID NO: 6-9 amino acid sequences, can elicit animmune response to about 97% of the 400 known Tat sequences of thecommon B subtypes of HIV-1 and with Tat proteins of all 18 non-B subtypeHIV-1 that have been sequenced (courtesy of Dr. Esther Guzman, LosAlamos NIAID HIV database; GenBank database).

[0066] B. Epitope II Immunogenic Compositions

[0067] In another embodiment, the present invention provides acomposition comprising at least one Epitope II amino acid sequence. ThisEpitope II sequence elicits a specific humoral immune response (for thepurpose of this invention) in a mammal exposed to the Epitope IIsequence in vivo. Epitope II defines peptides of the formulaR3-Lys-X-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys-R4, wherein X is Gly (70%)or Ala (30%). This sequence is highly conserved. The immunogen in whichX is Gly induces antibodies cross-reactive with the sequence in which Xis Ala.

[0068] The N terminal R3 may represent the hydrogen on the unmodified Nterminal amino acid Lys, or R3 may be a lower alkyl, or a loweralkanoyl, such as an acetyl group, substituent on the Lys. R3 may alsoinclude a sequence of between 1 to about 5 amino acids, optionallysubstituted with a lower alkyl or lower alkanoyl. The C terminal R4 mayrepresent the free hydroxyl of the C terminal amino acid Lys, or R4 maybe an amide on that C terminal amino acid. R4 may optionally be asequence of one or up to about 5 additional amino acids, optionallysubstituted with an amide. The presently preferred immunogen for EpitopeII is -Lys-Gly-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys- (amino acids 41-51of SEQ ID NO: 1). This would react/cross-react with greater than 96% ofknown HIV-1 Tat proteins.

[0069] Epitope II is poorly immunogenic when presented within othersequences. Thus, for optimal immunogenicity, this sequence is preparedas a synthetic peptide fused to, or coupled to, a carrier protein or asa multiple antigenic peptide, optionally coupled to carrier protein.Alternatively, Epitope II may be expressed as the C terminal sequence ofa recombinant protein, which is optionally fused in frame to a carrierprotein at its amino terminal sequence. In a composition of thisinvention, an Epitope II peptide is preferably presented alone or incombination with one or more Epitope I peptides. Other compositions mayemploy one or more Epitope III or IV peptides.

[0070] C. Epitope III Immunogenic Compositions

[0071] In another embodiment, the present invention provides acomposition comprising at least one, and preferably two or more EpitopeIII amino acid sequences. These Epitope III sequences elicit a specifichumoral immune response (for the purpose of this invention) in a mammalexposed to the Epitope III sequences in vivo. This epitope showsconsiderable more variation than Epitopes I and II. These Epitope IIIimmunogenic peptides and polypeptides are derived from Tat variantprotein sequences corresponding to amino acids 56-62 of SEQ ID NO: 1.Epitope III defines peptides of the formula. R5-Arg-Arg-X-Z-A-Y-Ser-R6(SEQ ID NO: 38), wherein X may be Ala, Pro, Ser or Gln; Y may be Asp,Asn, Gly or Ser; Z may be Pro or His; and A may be Gln or Pro. TheEpitope III immunogens in which X is Ala induce antibodies thatcross-react with the other position X variants. Epitope III immunogenscontaining Asp in position Y induce antibodies that cross-react with theother position Y variants. The three most common variants for positionsZ and A are -Pro-Gln- (61%), -Pro-Pro- (8%) and -His-Gln- (8%).Antibodies induced by these three immunogens do not cross-react with theothers so that three immunogens would need to be used to cover thesevariants (77%).

[0072] According to the formula of Epitope III above, the seven aminoresidues which form the minimum reactive Epitope III sequences, may beflanked by other amino acids, so that the entire Epitope III sequencemay be between 7 and about 15 amino acids in length. As indicated inExample 3 below, the identity of the flanking amino acids is notessential to the biological function of the Epitope III immunogen. Inparticular additional amino acids on the N-terminus of Epitope IIIsequences do not affect immunogenicity. The N terminal R5 may optionallyrepresent the hydrogen on the N-terminal Arg, or R5 is a lower alkyl oralkanoyl, such as an acetyl group, substituent on the N-terminal Arg.Alternatively, R5 is a sequence of between 1 to about 3 amino acids,optionally substituted with a lower alkyl or lower alkanoyl. In apreferred embodiment R5 is -Gln-Arg-, optionally modified as above,which improves the immunogenicity of the Epitope. The C terminal R6represents either the free hydroxyl on the C terminal amino acid or anamide substituent on the C terminal amino acid, because any C-terminalextension impairs immunogenicity.

[0073] Epitope III immunogens which can be present in compositions ofthis invention can include at least one copy of at least one of thefollowing preferred Epitope III amino acid sequences:R5-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser-R6,R5-Gln-Arg-Arg-Arg-Ala-His-Gln-Asp-Ser-R6, andR5-Gln-Arg-Arg-Arg-Ala-Pro-Pro-Asp-Ser-R6, optionally modified as above(SEQ ID NOS: 20 through 22, respectively).

[0074] Still other optional immunogenic sequences which may be includedin the Epitope III compositions includeR5-Arg-Arg-Pro-Pro-Gln-Asp-Asn-R6, R5-Arg-Arg-Ala-Pro-Gln-Asp-Arg-R6;R5-Arg-Gly-Ala-Pro-Gln-Asp-Ser-R6; R5-Arg-Arg-Ala-Pro-Glu-Asp-Ser-R6; orR5-Arg-Arg-Ala-Ser-Gln-Asp-Ser-R6 (SEQ ID NOS: 23 through 27,respectively). As can be determined from review of the examples below,the inclusion of these Epitope III peptides in compositions of theinvention can induce antibodies that react with rare Tat proteins ofHIV-1 which are not cross-reactive with, or do not have a sufficientlystrong cross-reactivity to, antibodies induced by the preferred EpitopeIII immunogens.

[0075] As described in more detail below, the Epitope III peptides orpolypeptides are poorly immunogenic when presented within othersequences. Although the Epitope III sequences may be preparedrecombinantly, for optimal immunogenicity, these sequences would beprepared synthetically and coupled to a carrier protein, or as multipleantigenic peptides, optionally coupled to carrier protein.Alternatively, Epitope III may be expressed as the C terminal sequenceof a recombinant protein, which is optionally fused in frame to acarrier protein at its amino terminal sequence. Compositions of thisinvention would preferably contain three or more different Epitope IIIimmunogens, optionally with at least one Epitope I immunogen, andoptionally with one or more Epitope II or Epitope IV immunogens.

[0076] D. Epitope IV Immunogenic Compositions

[0077] In another embodiment, the present invention provides acomposition comprising at least one, and preferably two or more EpitopeIV amino acid sequences. These Epitope IV sequences elicit a specifichumoral immune response (for the purpose of this invention) in a mammalexposed to the Epitope IV sequences in vivo. The Epitope IV immunogenicpeptides and polypeptides are derived from Tat variant protein sequencescorresponding to amino acids 62-72 of SEQ ID NO: 1, including aC-terminal Pro from Exon 2 of HIV-1 Tat. Epitope IV defines peptides ofthe formula: R7-Ser-Gln-X-His-Gln-Y-Ser-Leu-Ser-Lys-Gln-Pro-R8 (SEQ IDNO: 39), wherein X may be Asn or Thr; and Y may be Ala or Val. Theimmunogen in which X is Thr induces antibodies that cross-react with theimmunogen in which X is Asn. The immunogen in which Y is Val induceantibodies that do not cross-react with the peptides in which Y is Ala.However, the peptides containing Ala in position Y induce antibodiesthat cross-react with peptides for Epitope IV in which Y is Val. Thusthe optimal Epitope IV immunogen is Ser-Gln-Thr-His-Gln-Ala-Ser-Leu-SerLys-Gln-Pro (SEQ ID NO: 40) and this induces antibodiesreactive/cross-reactive with 64% of known HIV-1 Tat proteins.

[0078] According to the formula of Epitope IV above, the twelve aminoresidues which form the minimum reactive Epitope IV sequences, may beflanked by a few other amino acids, so that the entire Epitope IVsequence may be between 12 and about 18 amino acids in length. The Nterminal R7 may represent the hydrogen of the N terminal amino acid, ora lower alkyl or alkanoyl, such as an acetyl group, substituent on the Nterminal amino acid. Although N-terminal extension markedly inhibitsimmunogenicity, the R7 may also be a sequence of between 1 to about 3amino acids, optionally substituted with a lower alkyl or loweralkanoyl. The C terminal R8 may represent the free hydroxyl on the Cterminal amino acid, or an amide substituent on the C terminal aminoacid, or R8 may be a sequence of one or up to about 3 additional aminoacids, optionally substituted with an amide. Additionally, theC-terminal Pro, which is an important component of the epitope, isencoded by Exon 2 of Tat. Thus antibodies to Epitope IV would be poorlyreactive with non-spliced Tat Exon 1 protein.

[0079] This Epitope IV sequence is poorly immunogenic when presentedwithin other sequences, Thus, for optimal immunogenicity, this sequencewould be prepared as a synthetic peptide coupled to carrier protein oras a multiple antigenic peptide, optionally coupled to carrier protein.Compositions of this invention would preferably contain two or moredifferent Epitope IV immunogens, optionally with at least one Epitope Iimmunogen, and optionally with one or more Epitope II or Epitope IIIimmunogens.

[0080] E. Compositions Containing Multiple Epitopes

[0081] While the amino acid sequences of Epitope I, II, III and IV andoptional immunogens identified herein were obtained by rigorous analysisof over 400 known Tat sequences of HIV-1, it should be understood by oneof skill in the art that similar compositions may be obtained, followingthe teachings of this invention, from the study of further Tat proteins,the nucleic acid sequences encoding them, and fragments thereof fromnewly isolated Tat proteins of HIV-1 subtype B, or from Tat proteins ofthe other subtypes, or from other HIV strains.

[0082] Thus, the compositions of this invention, i.e., thepeptide/polypeptides containing the above-identified amino acidsequences, when provided to a human subject, are useful in theimmunologic interdiction of extracellular Tat proteins of most HIV-1strains. These compositions function to critically reduce explosivemultiplication of the virus and permit effective immune control of thevirus.

[0083] The immunogens for each Epitope are preferably designed to induceantibodies reactive with the highest proportion of naturally occurringvariants of each epitope. For an epitope such as Epitope I, multiplecopies of an immunogen could be incorporated in a synthetic orrecombinant immunogen to enhance the immunogenicity and produce highertiter antibodies. Furthermore, immunogens for two or more epitopes couldbe combined to extend coverage, since variations in sequence of eachepitope occur independently. For example, combining an Epitope Iimmunogen(s) (95%) with an Epitope II immunogen (96%) would result inantibodies in immunologically responsive subjects reactive with 99.8% ofknown HIV-1 Tat proteins. Thus, as one example, a composition of thisinvention contains one Epitope I (underlined)-Epitope II(double-underlined) fused peptide immunogen such asCys-Glu-Pro-Val-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Lys-Glu-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys-amide(SEQ ID NO: 67), coupled to carrier protein attached to Epitope I, orthe same peptide (less the N-terminal Cys for coupling), synthesized asa multiple antigenic peptide, optionally coupled to a carrier protein.Alternatively, mixtures of two or more immunogens could be used asfollows.

[0084] The Epitope I immunogens, with or without any Epitope II, III orIV or other optional immunogens, may be prepared and used in immunogeniccompositions in a variety of forms, for example, chemically synthesizedor as recombinant peptides, polypeptides, proteins, fusion proteins orfused peptides.

[0085] 1. Synthetic Peptide/Protein Coupled to a Carrier

[0086] As one embodiment, a composition of the present invention may bea synthetic peptide, containing single or multiple copies of the same ordifferent Epitope I immunogen amino acid sequences and/or EpitopeII/III/IV immunogenic amino acid sequences, and optionally amino acidsequences of the optional immunogens, coupled to a selected carrierprotein. In this embodiment of a composition of this invention, multipleabove-described Epitope I amino acid sequences with or without flankingsequences, may be combined sequentially in a polypeptide and coupled tothe same carrier. Alternatively, the Epitope I, II, III, or IVimmunogens, may be coupled individually as peptides to the same ordifferent carrier proteins, and the resulting immunogen-carrierconstructs mixed together to form a single composition.

[0087] For this embodiment, the carrier protein is desirably a proteinor other molecule which can enhance the immunogenicity of the selectedimmunogen. Such a carrier may be a larger molecule which has anadjuvanting effect. Exemplary conventional protein carriers include,without limitation, E. coli DnaK protein, galactokinase (galK, whichcatalyzes the first step of galactose metabolism in bacteria),ubiquitin, α-mating factor, β-galactosidase, and influenza NS-1 protein.Toxoids (i.e., the sequence which encodes the naturally occurring toxin,with sufficient modifications to eliminate its toxic activity) such asdiphtheria toxoid and tetanus toxoid may also be employed as carriers.Similarly a variety of bacterial heat shock proteins, e.g.,mycobacterial hsp-70 may be used Glutathione reductase (GST) is anotheruseful carrier. One of skill in the art can readily select anappropriate carrier.

[0088] In particularly desirable immunogen-carrier protein construct,one or more epitope immunogen and optional immunogenpeptides/polypeptides may be covalently linked to a mycobacterial E.coli heat shock protein 70 (hsp70) (K. Suzue et al, J. Immunol., 156:873(1996)). In another desirable embodiment, the composition is formed bycovalently linking the immunogen-containing peptide or polypeptidesequences to diphtheria toxoid.

[0089] 2. Multiple Antigenic Peptide

[0090] In yet another embodiment, the peptides or polypeptide epitopeimmunogens and any selected optional immunogens may be in the form of amultiple antigenic peptide (“MAP”, also referred to as an octamericlysine core peptide) construct. Such a construct may be designedemploying the MAP system described by Tam, Proc. Natl. Acad. Sci. USA,85:5409-5413 (1988). This system makes use of a core matrix of lysineresidues onto which multiple copies of the same Epitope I or optionalimmunogens of the invention are synthesized as described (D. Posnett etal., J. Biol. Chem., 263(4):1719-1725 (1988); J. Tam, “ChemicallyDefined Synthetic Immunogens and Vaccines by the Multiple AntigenPeptide Approach”, Vaccine Research and Developments, Vol. 1, ed. W.Koff and H. Six, pp. 51-87 (Marcel Deblau, Inc., New York 1992)). EachMAP contains multiple copies of only one peptide. Therefore, e.g., anepitope composition of this invention can include a MAP in which thepeptide or polypeptide epitope immunogen attached to the lysine corecontains one or sequential repeats of the four amino acid sequences (SEQID NOS: 6-9) identified above. Multiple different MAPs may be employedto obtain any desired combination of Epitope I, II, III or IV sequences.Preferably these MAP constructs are associated with other T cellstimulatory sequences, or as pharmaceutical compositions, administeredin conjunction with T cell stimulatory agents, such as known adjuvants.

[0091] 3. Spacers

[0092] In either of the above compositions, e g, aspeptide/polypeptide-carrier constructs or MAPs, each peptide/polypeptideimmunogen, or each amino acid sequence in the immunogen, may beoptionally separated by optional amino acid sequences called “spacers”.Spacers are sequences of between 1 to about 4 amino acids which areinterposed between two sequences to permit linkage therebetween withoutadversely effecting the three dimensional structure of the immunogen.Spacers may also contain restriction endonuclease cleavage sites toenable separation of the sequences, where desired Suitable spacers orlinkers are known and may be readily designed and selected by one ofskill in the art. Preferred spacers are sequences containing Gly and/orSer amino acids.

[0093] F. Nucleic Acid Compositions of the Invention, Including aSynthetic Gene

[0094] Other embodiments of this invention include nucleic acidsequences, which encode the above-described peptide/polypeptidecompositions, including the peptide and polypeptide immunogens of thecompositions described above, including those peptides and polypeptidesfused to carrier proteins. The nucleic acid sequences may also includesequences encoding the carrier proteins.

[0095] Thus, one preferred embodiment of the invention is a “syntheticgene” which encodes sequentially for one or more Epitope I immunogenicpeptides/polypeptide. The synthetic gene preferably encodes two, threeor all four Epitope I amino acid sequences (SEQ ID NOS: 6 through 9,respectively):

[0096] R1-Val-Asp-Pro-Arg-Leu-Glu-Pro-R2;

[0097] R1-Val-Asp-Pro-Lys-Leu-Glu-Pro-R2;

[0098] R1-Val-Asp-Pro-Ser-Leu-Glu-Pro-R2; and

[0099] R1-Val-Asp-Pro-Asn-Leu-Glu-Pro-R2.

[0100] The synthetic gene can also encode any selection of the optionalimmunogens identified above, and may include an Epitope II or IIIimmunogen provided that the Epitope II or III peptide is fused to the Cterminus of the Epitope I sequence and not further modified on its own Cterminus. The synthetic gene may encode multiple copies of the sameamino acid sequence, copies of multiple different immunogens or aminoacid sequences, or multiple copies of multiple different immunogens oramino acid sequences. The synthetic gene may encode the selected aminoacid sequences in an open reading frame with, or fused to, a nucleicacid sequence encoding a carrier protein. A further characteristic ofthe synthetic gene may be that it encodes a spacer between each sequenceencoding an immunogen and/or between the sequence encoding an immunogenand the sequence encoding the carrier protein.

[0101] The synthetic gene of the present invention may also be part of asynthetic or recombinant molecule. The synthetic molecule may be anucleic acid construct, such as a vector or plasmid which contains thesynthetic gene encoding the protein, peptide, polypeptide, fusionprotein or fusion peptide under the operative control of nucleic acidsequences encoding regulatory elements such as promoters, terminationsignals, and the like. Such synthetic molecules may be used to producethe polypeptide/peptide immunogen compositions recombinantly.

[0102] The synthetic gene or synthetic molecules can be prepared by theuse of chemical synthesis methods or preferably, by recombinanttechniques. For example, the synthetic gene or molecules may containcertain preference codons for the species of the indicated host cell.

[0103] The synthetic gene or molecules, preferably in the form of DNA,may be used in a variety of ways. For example, these synthetic nucleicacid sequences may be employed to express the peptide/polypeptides ofthe invention in vitro in a host cell culture. The expressed immunogens,after suitable purification, may then be incorporated into apharmaceutical reagent or vaccine.

[0104] Alternatively, the synthetic gene or synthetic molecule of thisinvention may be administered directly into a mammalian, preferablyhuman subject, as so-called ‘naked DNA’ to express the protein/peptideimmunogen in vivo in a patient. See, e.g., J. Cohen, Science,259:1691-1692 (Mar. 19, 1993), E Fynan et al., Proc. Natl. Acad. Sci.,USA, 90:11478-11482 (December 1993); and J. A. Wolff et al.,Biotechniques, 11:474-485 (1991), all incorporated by reference herein.The synthetic molecule, e.g., a vector or plasmid, may be used fordirect injection into the mammalian host. This results in expression ofthe protein by host cells and subsequent presentation to the immunesystem to induce antibody formation in vivo.

[0105] G. Microorganisms that Express the Synthetic Gene

[0106] In still another aspect of the present invention, the syntheticgenes or molecules of this invention may be incorporated into anon-pathogenic microorganism. The resulting microorganism, whenadministered to a mammalian host expresses and multiplies the expressedcompositions of this invention in vivo to induce specific antibodyformation. For example, non-pathogenic recombinant viruses or commensalbacterium which carry the compositions or synthetic genes of thisinvention and are useful for administration to a mammalian patient maybe prepared by use of conventional methodology and selected from amongknown non-pathogenic microorganisms.

[0107] Among commensal bacterium which may be useful for exogenousdelivery of the synthetic molecule to the patient, and/or for carryingthe synthetic gene into the patient in vivo, include, for example,various strains of Streptococcus, e.g., S. gordonii, or E. coli,Bacillus, Streptomyces, and Saccharomyces.

[0108] Suitable non-pathogenic viruses which may be engineered to carrythe synthetic gene into the cells of the host include poxviruses, suchas vaccinia, adenovirus, canarypox, retroviruses and the like. A numberof such non-pathogenic viruses are commonly used for human gene therapy,and as carrier for other vaccine agents, and are known and selectable byone of skill in the art.

[0109] H. Preparation or Manufacture of Compositions of the Invention

[0110] The compositions of the invention, and the individualpolypeptides/peptides containing the Epitope I, II, III or Epitope IV oroptional immunogens of this invention, the synthetic genes, andsynthetic molecules of the invention, may be prepared conventionally byresort to known chemical synthesis techniques, such as described byMerrifield, J. Amer. Chem. Soc., 85:2149-2154 (1963). Alternatively, thecompositions of this invention may be prepared by known recombinant DNAtechniques by cloning and expressing within a host microorganism or cella DNA fragment carrying a sequence encoding a peptide/polypeptidecontaining an Epitope I and/or optional immunogen and optional carrierprotein. Coding sequences for the Epitope I and optional immunogens canbe prepared synthetically (W. P. C. Stemmer et al, Gene, 164:49 (1995)or can be derived from viral RNA by known techniques, or from availablecDNA-containing plasmids.

[0111] Combinations of these techniques may be used, such as forproduction of the synthetic gene, which may require assembly ofsequential immunogens by conventional molecular biology techniques, andsite-directed mutagenesis to provide desired sequences of immunogens.The product of the synthetic gene is then produced recombinantly. All ofthese manipulations may be performed by conventional methodology.

[0112] Systems for cloning and expressing the peptide/polypeptidecompositions of this invention using the synthetic genes or molecules,include various microorganisms and cells which are well known inrecombinant technology. These include, for example, various strains ofE. coli, Bacillus, Streptomyces, and Saccharomyces, as well asmammalian, yeast and insect cells. Suitable vectors therefor are knownand available from private and public laboratories and depositories andfrom commercial vendors. Currently, the most preferred host is amammalian cell such as Chinese Hamster ovary cells (CHO) or COS-1 cells.These hosts may be used in connection with poxvirus vectors, such asvaccinia or swinepox. The selection of other suitable host cells andmethods for transformation, culture, amplification, screening andproduct production and purification can be performed by one of skill inthe art by reference to known techniques. See, e.g., Gething andSambrook, Nature, 293:620-625 (1981).

[0113] Another preferred system includes the baculovirus expressionsystem and vectors.

[0114] When produced by conventional recombinant means, the compositionsof this invention, i.e., the polypeptide/peptides containing theindicated copies of the Epitope I immunogens and optional immunogens maybe isolated either from the cellular contents by conventional lysistechniques or from cell medium by conventional methods, such aschromatography. See, e g., Sambrook et al., Molecular Cloning. ALaboratory Manual., 2d Edit., Cold Spring Harbor Laboratory, New York(1989).

[0115] Suitable plasmid and viral vectors used either for production ofthe peptide/polypeptide components as DNA vaccines are well known tothose of skill in the art and are not a limitation of the presentinvention. See, Sambrook et al., cited above and the references above toproduction of the protein. See, also International Patent PublicationNo. WO94/01139, published Jan. 20, 1994

[0116] Briefly, the DNA encoding the selected peptide/polypeptide isinserted into a vector or plasmid which contains other optional flankingsequences, a promoter, an mRNA leader sequence, an initiation site andother regulatory sequences capable of directing the multiplication andexpression of that sequence in vivo or in vitro. These vectors permitinfection of patient's cells and expression of the synthetic genesequence in vivo or expression of it as a protein/peptide or fusionprotein/peptide in vitro.

[0117] The resulting composition may be formulated into a Epitope Icomposition with any number of optional immunogens and screened forefficacy by in vivo assays. Such assays employ immunization of ananimal, e.g., a rabbit or a simian, with the composition, and evaluationof titers of antibody to the Tat proteins of HIV-1 or to syntheticdetector peptides corresponding to variant Tat sequences (as shown inthe examples below).

[0118] I. Antibody Compositions of the Invention

[0119] An isolated mammalian antibody composition which is directedagainst a peptide or polypeptide of the invention, as described above,is also an aspect of this invention. Such polyclonal antibodycompositions are produced by immunizing a mammal with apeptide/polypeptide composition containing an assortment of Epitope I,II, III, and/or IV immunogens and optional immunogens, as describedabove. Suitable mammals include primates, such as monkeys; smallerlaboratory animals, such as rabbits and mice, as well as larger animals,such as horse, sheep, and cows. Such antibodies may also be produced intransgenic animals. However, a desirable host for raising polyclonalantibodies to a composition of this invention includes humans.

[0120] The polyclonal antibodies raised in the mammal exposed to thecomposition are isolated and purified from the plasma or serum of theimmunized mammal by conventional techniques. Conventional harvestingtechniques can include plasmapheresis, among others.

[0121] Such polyclonal antibody compositions may themselves be employedas pharmaceutical compositions of this invention Alternatively, otherforms of antibodies may be developed using conventional techniques,including monoclonal antibodies, chimeric antibodies, humanizedantibodies and fully human antibodies. See, e.g., Harlow et al.,Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, (1988);Queen et al., Proc. Nat'l. Acad. Sci. USA, 86:10029-10032 (1989);Hodgson et al., Bio/Technology, 9:421 (1991); International PCTApplication PCT/GB91/01554, Publication No. WO92/04381 and InternationalPCT Application PCT/GB93/00725, Publication No. WO93/20210). Otheranti-Tat antibodies may be developed by screening hybridomas orcombinatorial libraries, or antibody phage displays (W. D. Huse et al.,Science, 246:1275-1281 (1988)) using the polyclonal or monoclonalantibodies produced according to this invention and the amino acidsequences of the Epitope I, II, III, IV or optional immunogens.

[0122] These antibody compositions bind to greater than 95%, andpreferably greater than 99% of known Tat protein variants of HIV-1, andprevent the Tat proteins from supporting further HIV-1 multiplication.Thus, these antibodies are useful in pharmaceutical methods andformulations described below.

[0123] J. Pharmaceutical Compositions of the Invention

[0124] As another aspect of this invention, a pharmaceutical compositionuseful for inducing antibodies that react with greater than 95%,preferably greater than 99%, of known HIV-1 Tat proteins and impair themultiplication of HIV-1 can comprise as its active agents, one or moreof the peptides or polypeptides of Epitope I, II, III, or IV. Severaldesirable compositions include the following above-described components:

[0125] (a) a peptide/polypeptide immunogen which contains at least one,and preferably all four of the Epitope I amino acid sequences SEQ IDNOS: 6-9;

[0126] (b) a peptide/polypeptide immunogen which contains at least oneof the Epitope II amino acid sequences;

[0127] (c) a peptide/polypeptide immunogen which contains at least one,and preferably three Epitope III amino acid sequences;

[0128] (d) a peptide/polypeptide immunogen which contains at least oneEpitope IV amino acid sequence;

[0129] (e) a synthetic gene encoding one or more of Epitope I, EpitopeII, Epitope III, or Epitope IV sequences as described above;

[0130] (f) a synthetic molecule described above;

[0131] (g) a recombinant virus carrying the synthetic gene or molecule;and

[0132] (h) a commensal bacterial carrying the synthetic gene ormolecule.

[0133] The selected active component(s) is present in a pharmaceuticallyacceptable carrier, and the composition may contain additionalingredients. Pharmaceutical formulations containing the compositions ofthis invention may contain other active agents, such as T cellstimulatory agents for the MAPs, adjuvants and immunostimulatorycytokines, such as IL-12 and other well-known cytokines, for theprotein/peptide compositions. All of these pharmaceutical compositionscan operate to lower the viral levels of a mammal.

[0134] As pharmaceutical compositions, these compositions comprisingEpitope I and/or II, and/or III, and/or IV amino acid sequences withoptional immunogenic amino acid sequences are admixed with apharmaceutically acceptable vehicle suitable for administration as aprotein composition for prophylaxis or treatment of virus infections.These proteins may be combined in a single pharmaceutical preparationfor administration. Suitable pharmaceutically acceptable carriers foruse in an immunogenic proteinaceous composition of the invention arewell known to those of skill in the art. Such carriers include, forexample, saline, buffered saline, a selected adjuvant, such as aqueoussuspensions of aluminum and magnesium hydroxides, liposomes, oil inwater emulsions and others. Suitable adjuvants may also be employed inthe protein-containing compositions of this invention. The presentinvention is not limited by the selection of the carrier or adjuvant.

[0135] Suitable vehicles for direct DNA, plasmid nucleic acid, orrecombinant vector administration include, without limitation, saline,or sucrose, protamine, polybrene, polylysine, polycations, proteins,CaPO₄ or spermidine. See e.g, International Patent Publication No.WO94/01139 and the references cited above.

[0136] The peptide/polypeptide compositions and synthetic genes ormolecules in vivo are capable of eliciting in an immunized host mammal,e.g., a human, an immune response capable of interdicting greater thanabout 95 to about 99% of known extracellular Tat protein variants fromHIV-1 and thereby lowering the viral levels.

[0137] Yet another pharmaceutical composition useful for impairing themultiplication of HIV-1 comprises an antibody composition as describedin detail above. In a pharmaceutical composition, the antibodies may becarried in a saline solution or other suitable carrier. The antibodycompositions are capable of providing an immediate, exogenously providedinterdiction of Tat.

[0138] The preparation of these pharmaceutically acceptablecompositions, from the above-described components, having appropriate pHisotonicity, stability and other conventional characteristics is withinthe skill of the art.

[0139] K. Method of the Invention—Impairing Multiplication of HIV-1

[0140] According to the present invention, a method for reducing theviral levels of HIV-1 involves exposing a human to the Tatantibody-inducing pharmaceutical compositions described above, activelyinducing antibodies that react with greater than 95%, preferably greaterthan 99%, of known HIV-1 Tat proteins, and impairing the multiplicationof the virus in vivo. This method is appropriate for an HIV-1 infectedsubject with a competent immune system, or an uninfected or recentlyinfected subject The method induces antibodies which react with HIV-1Tat proteins, which antibodies reduce viral multiplication during anyinitial acute infection with HIV-1 and minimize chronic viremia leadingto AIDS. This method also lowers chronic viral multiplication ininfected subjects, again minimizing progression to AIDS.

[0141] In one embodiment, the pharmaceutical compositions may betherapeutically administered to an HIV-1 infected human with a competentimmune system for treatment or control of viral infection. Such aninfected human may be asymptomatic. In a similar embodiment, thepharmaceutical compositions may be administered to an uninfected humanfor prophylaxis.

[0142] In these two instances, the pharmaceutical compositionspreferably contain the peptide/polypeptide compositions, the syntheticgenes or molecules, the recombinant virus or the commensal recombinantbacterium. Each of these active components of the pharmaceuticalcomposition actively induces in the exposed human the formation ofanti-Tat antibodies which block the transfer of Tat from infected cellsto other infected or uninfected cells. This action reduces themultiplicity of infection and blocks the burst of HIV-1 viral expansion,and thus lowers viral levels. In already infected patients, this methodof reduction of viral levels can reduce chronic viremia and progressionto AIDS. In uninfected humans, this administration of the compositionsof the invention can reduce acute infection and thus minimize chronicviremia leading to progression to AIDS.

[0143] Yet another aspect of the invention is a method for reducing theviral levels of HIV-1 by administering to a human, who is incapable ofmounting an effective or rapid immune response to infection with HIV-1,a pharmaceutical composition containing the antibody compositionsdescribed above. The method can involve chronically administering thecomposition. Among such patients suitable for treatment with this methodare HIV-1 infected patients who are immunocompromised by disease andunable to mount a strong immune response. In later stages of HIVinfection, the likelihood of generating effective titers of antibodiesis less, due to the immune impairment associated with the disease. Alsoamong such patients are HIV-1 infected pregnant women, neonates ofinfected mothers, and unimmunized patients with putative exposure (e.g.,a human who has been inadvertently “stuck” with a needle used by anHIV-1 infected human).

[0144] For such patients, the method of the invention preferably employsas the pharmaceutical composition the antibody composition of theinvention, which is a polyclonal antibody composition prepared in othermammals, preferably normal humans. Alternatively, the other forms ofantibody described above may be employed. These antibody compositionsare administered as passive immunotherapy to inhibit viralmultiplication and lower the viral load. The exogenous antibodies whichreact with greater than 95%, preferably greater than 99%, of known Tatproteins from HIV-1 provide in the patient an immediate interdiction ofthe transfer of Tat from virally infected cells to other infected oruninfected cells. According to this method, the patient may bechronically treated with the antibody composition for a long treatmentregimen.

[0145] In each of the above-described methods, these compositions of thepresent invention are administered by an appropriate route, e.g., by thesubcutaneous, oral, intravenous, intraperitoneal, intramuscular, nasal,or inhalation routes. The presently preferred route of administration isintramuscular for the immunizing (active induction) compositions andintravenous or intramuscular for the antibody (passive therapy)compositions. The recombinant viral vectors or naked DNA is preferablyadministered i.m.; however, other certain recombinant viral vectorsand/or live commensal bacteria may be delivered orally.

[0146] The amount of the protein, peptide or nucleic acid sequences ofthe invention present in each vaccine dose is selected with regard toconsideration of the patient's age, weight, sex, general physicalcondition and the like. The amount of active component required toinduce an immune response, preferably a protective response, or producean exogenous effect in the patient without significant adverse sideeffects varies depending upon the pharmaceutical composition employedand the optional presence of an adjuvant (for the protein-containingcompositions).

[0147] Generally, for the compositions containing protein/peptide,fusion protein, MAP or coupled protein, or antibody composition, eachdose will comprise between about 50 μg to about 2 mg of thepeptide/polypeptide immunogens per mL of a sterile solution. A morepreferred dosage may be about 500 μg of immunogen. Other dosage rangesmay also be contemplated by one of skill in the art. Initial doses maybe optionally followed by repeated boosts, where desirable.

[0148] The antibody compositions of the present invention can beemployed in chronic treatments for subjects at risk of acute infectiondue to needle sticks or maternal infection. A dosage frequency for such“acute” infections may range from daily dosages to once or twice a weeki.v. or i.m., for a duration of about 6 weeks. The antibody compositionsof the present invention can also be employed in chronic treatments forinfected patients, or patients with advanced HIV. In infected patients,the frequency of chronic administration may range from daily dosages toonce or twice a week i v. or i.m., and may depend upon the half-life ofthe immunogen (e.g., about 7-21 days). However, the duration of chronictreatment for such infected patients is anticipated to be an indefinite,but prolonged period.

[0149] Alternatively, compositions of this invention may be designed fordirect administration of synthetic genes or molecules of this inventionas “naked DNA”. As with the protein immunogenic compositions, theamounts of components in the DNA and vector compositions and the mode ofadministration, e.g., injection or intranasal, may be selected andadjusted by one of skill in the art. Generally, each dose will comprisebetween about 50 μg to about 1 mg of immunogen-encoding DNA per mL of asterile solution.

[0150] For recombinant viruses containing the synthetic genes ormolecules, the doses may range from about 20 to about 50 ml of salinesolution containing concentrations of from about 1×10⁷ to 1×10¹⁰ pfu/mlrecombinant virus of the present invention A preferred human dosage isabout 20 ml saline solution at the above concentrations. However, it isunderstood that one of skill in the art may alter such dosages dependingupon the identity of the recombinant virus and the make-up of theimmunogen that it is delivering to the host.

[0151] The amounts of the commensal bacteria carrying the synthetic geneor molecules to be delivered to the patient will generally range betweenabout 1×10³ to about 1×10¹² cells/kg. These dosages, will of course, bealtered by one of skill in the art depending upon the bacterium beingused and the particular composition containing Epitope I, or Epitope IIor Epitope III or Epitope IV and optional immunogens being delivered bythe live bacterium.

[0152] Thus, the compositions of this invention are designed to retardor minimize infection by the selected virus of an uninfected mammal,e.g., human Such compositions thus have utility as vaccines. Anti-Tatprotein antibodies are not reactive with the HIV-1 proteins used indiagnostic assays to detect seroconversion after infection. Thus,subjects treated with the compositions of this invention would not bestigmatized with false-positive tests for HIV-1 infection, and it wouldremain possible to detect seroconversion if treated subjects did becomeinfected with HIV-1.

[0153] Providing a mammal with the compositions of this invention,whether as a protein/peptide-containing composition or by administrationof a novel nucleic acid sequence encoding the immunogen, affords aradically different strategy for AIDS vaccination because it permits thelowering of viral levels by biological interdiction of greater thanabout 95%, and preferably greater than about 99%, of known Tat proteinvariants of HIV-1, lowering multiplication of HIV-1.

[0154] The use of the Tat immunogen-containing compositions has aparticularly desirable advantage in contrast to other treatments andprophylactic methods employed against such viruses. Because interdictionof the Tat protein extracellularly inhibits the multiplication of allHIV quasi-species or strains indiscriminately, it does not create aselective pressure on the parent virus itself for selection of mutantvirus variants. Thus, blocking the uptake of Tat protein by thepatient's cells not only reduces the level of viremia, but does so in amanner that precludes the selection of “escape variants”.

[0155] Additionally, the invention comprises a method of activelytreating asymptomatic HIV-1 infected subjects with viremia, since duringthe course of the disease, extracellular Tat protein likely contributesto the persistent infection and immune abnormalities that are present atthis stage of HIV-1 infection. Interdiction of extracellular Tat proteinby antibodies induced by immunization according to this invention canreduce viremia with more effective immune control, and result in delayor prevention of progression to AIDS.

[0156] The mechanism of the present invention as described above isuseful in impeding the course of viral infection and producing desirableclinical results. More specifically, the compositions of this inventionare capable of reducing viremia in patients already infected with thevirus by blocking further uptake of the Tat protein by uninfected cells.The compositions of the present invention, used either alone or inconjunction with other therapeutic regimens for HIV infected patients,are anticipated to assist in the reduction of viremia and prevention ofclinical deterioration.

[0157] For such therapeutic uses, the formulations and modes ofadministration are substantially identical to those describedspecifically above and may be administered concurrently orsimultaneously with other conventional therapeutics for the specificviral infection. For therapeutic use or prophylactic use, repeateddosages of the immunizing compositions may be desirable, such as ayearly booster or a booster at other intervals.

[0158] L. Diagnostic Kits of this Invention

[0159] The peptides and polypeptides described above can also beemployed as reagents of a kit useful for the measurement and detectionof titers and specificities of antibodies induced by vaccination withthe compositions described above. The kit of the invention can includeone or more peptides of Epitopes I through IV. In one embodiment, eachpeptide has on its N terminus the protein biotin and a spacer, e.g.,-Ser-Gly-Ser-Gly- (SEQ ID NO: 30). Alternatively, the peptide may haveon its C terminus a spacer, e g, -Gly-Ser-Gly-Ser- (SEQ ID NO: 90), andthe protein biocytin. These embodiments enable the peptides to be boundto an avidin-coated solid support, e.g., a plate or beads. Of course,other binding agents known to those of skill in the diagnostic assay artmay also be employed for the same purposes. Also provided in the kitare, labeled reagents which detect the binding of antibody to theimmobilized Epitope peptides, such as a goat anti-human immunoglobulinor the like. The label on the reagent may be selected from the manyknown diagnostic labels, such as radioactive compounds, fluorescentcompounds and proteins, colorimetric enzymes, etc. The kit thus alsocontains miscellaneous reagents and apparatus for reading labels, e.g.,certain substrates that interact with an enzymatic label to produce acolor signal, etc., apparatus, for taking blood samples, as well asappropriate vials and other diagnostic assay components. One of skill inthe art may also readily select other conventional diagnostic componentsfor this kit.

[0160] Such kits and reagents may be employed in a method for detectingthe titers and reactivity patterns of antibodies in subjects vaccinatedwith the compositions of this invention A method for determining thepresence and or titer of antibodies induced by immunization to a Tatimmunogen includes the steps of contacting a biological sample from animmunized subject, e.g., a body fluid, preferably blood, serum orplasma, but also possibly urine, saliva and other fluids or tissue, withone or more of the binding sequences of Epitope I, II, III or IV,preferably immobilized on a solid support, such as a plate or beads. TheEpitope I, II, III or IV binding sequences employed in this method maybe the minimal binding regions, unmodified. Therefore, such sequencesinclude -Val-Asp-Pro-Y-Leu-Glu-Pro- (SEQ ID NO: 86) or-Glu-Pro-Val-Asp-Pro-Y-Leu-Glu-Pro- (SEQ ID NO: 124), wherein Y isselected from the group consisting of Arg, Lys, Ser and Asn; and/or-Lys-X-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys- (SEQ ID NO: 87), wherein Xis selected from the group consisting of Gly or Ala; and/or-Arg-Arg-X-Z-A-Y-Ser- (SEQ ID NO 88), wherein X is selected from thegroup consisting of Ala, Pro, Ser and Gln; wherein Y is selected fromthe group consisting of Asp, Asn, Gly and Ser; wherein Z is selectedfrom the group consisting of Pro and His; wherein A is selected from thegroup consisting of Gln and Pro; and/or-Ser-Gln-X-His-Gln-Y-Ser-Leu-Ser-Lys-Gln-Pro- (SEQ ID NO: 89), wherein Xis selected from the group consisting of Asn and Thr; wherein Y isselected from the group consisting of Ala and Val.

[0161] Once the biological sample is exposed to the immobilized peptidesfor a sufficient time, the support is washed to eliminate any materialfrom the biological sample which is not bound to the peptides. Suchwashing steps are conventional in diagnostic assays, and performed withsaline. If antibodies to Epitopes I, or II, or III, or IV, or acombination thereof, were induced in the subject by the above-describedtreatment, the immobilized peptides have been bound with an antibodyfrom the biological sample. Thereafter, a labeled reagent is added tothe material on the support to detect the binding between the peptideson the solid support and antibody in said biological sample. Preferably,such a reagent is an anti-human immunoglobulin, such as goat anti-humanimmunoglobulin. The label is selected from among a wide array ofconventionally employed diagnostic labels, as discussed above. In oneembodiment, the label can be a colorimetric enzyme, which upon contactwith a substrate produces a detectable color signal. The presence and/orintensity of the color provides evidence of the induction of antibody inthe treated subject. This assay may be employed to determine theefficacy of the immunization, as well as to monitor immune status of apatient.

[0162] Again, the selection of particular assay steps, as well as avariety of detectable label systems, is well within the skill of theart. Such selection is routine and does not limit the present invention.

[0163] M. Advantages of the Invention

[0164] One of the advantages of the compositions of this invention isthe small number of immunogens required for inclusion into a compositionof this invention to cross-react with greater than 95 to greater than99% of known Tat protein variants of HIV-1 of the common B subtype. Asmentioned above, an immunogen of Epitope I in which Y was Lys (SEQ IDNO: 7) could suffice for full cross-reactivity to all four position Yvariants, and could be used alone in an immunogenic composition.Alternatively, as illustrated in the examples below, the Epitope Iimmunogenic composition containing all four Epitope I amino acidsequences cross-reacts with 387 of 399 Tat proteins of HIV-1 of thecommon B subtype, as well as with all 18 Tat protein sequences from lessfrequent non-B subtypes of HIV-1. Thus, a single composition may beusefully employed in protecting against or treating infection, caused bythe vast majority of HIV-1 strains that can be encountered.

[0165] Further, having identified the precise epitopes on Tat againstwhich binding is desired (i.e., AA2-10, AA41-51, or AA56-62 of SEQ IDNO: 1) new desirable Tat peptide immunogens from newly occurring HIV-1strains or newly discovered strains may be easily identified using themethods described herein, and included in the compositions. Thisflexibility enables the compositions of this invention to be usefulprophylactically against any new strain or strains of HIV-1 identifiedin the future. In view of the teachings herein, one of skill in the artis expected to be readily able to incorporate new combinations of Tatimmunogens (and the nucleic acid constructs encoding them) into thecompositions.

[0166] For example, the use of conventional techniques such as PCR andhigh density oligonucleotide arrays (M. J. Kozal et al, Nature Med.,2:753 (1996)) enables one of skill in the art to obtain the amino acidsequences of a large array of HIV-1 Tat proteins representing variantsof clinical isolates of HIV-1 strains and subtypes. Using suchtechniques permits determination of other variants of the HIV-1 Bsubtype as well as other subtypes in undeveloped countries, which havenot been so intensively studied to date. The determination of new Tatsequences will enable ready inclusion of the corresponding peptides asimmunogens into compositions of this invention, allowing the inductionof an antibody response against other rare Tat proteins of HIV-1.

[0167] Cross-reactivity studies with antibodies raised to syntheticpeptides corresponding to each Tat variant can be utilized to eliminatethe need for immunizing with Tat variants in which the sequence changesare immunologically silent, in that these peptides are strongly bound byantibodies to the consensus sequence or other variants.

[0168] The following examples illustrate preferred methods for preparingthe compositions of the invention and utilizing these compositions toinduce antibodies to Tat proteins of the virus in an immunized host.These examples are illustrative only and do not limit the scope of theinvention.

EXAMPLE 1 Immunological Studies on Minimal Tat Protein Amino AcidSequences Necessary for Binding to Antibody for Epitope I in HIV-1 TatProtein

[0169] A peptide corresponding to amino acids 4-16 of SEQ ID NO: 1illustrated in FIG. 1 was synthesized as described below. This sequenceis among the most frequent sequence representation at these positions in31 Tat protein sequences of the common B subtype reported in the NIAIDHIV database. This sequence was chosen as a putative immunogen, namedEpitope I.

[0170] A. Peptide Synthesis—Immunizing Peptides

[0171] The amino acid sequence of this immunogen-Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser- (SEQ ID NO: 28)was synthesized by solid phase methodology on polypropylene pegsaccording to the methods of H. M. Geysen et al., J. Immunol. Meth.,102:259 (1987), with an N-terminal cysteinyl being incorporated tofacilitate coupling to a carrier protein. The N-terminus was left as afree amine and the C-terminus was amidated in the immunizing peptidesfor most of the experiments for which data is reported in Table 2 belowImmunizing peptides were generally purified to greater than 95% purityby reverse phase HPLC, and purity was further confirmed by massspectometry (MS).

[0172] Immunizing peptides were covalently coupled to diphtheria toxoid(DT) carrier protein via the cysteinyl side chain by the method of A C.J. Lee et al., Molec. Immunol., 17:749 (1980), using a ratio of 6-8moles peptide per mole of diphtheria toxoid.

[0173] B. Peptide Synthesis—Detector Peptides

[0174] A peptide corresponding to the amino acid sequence of theimmunogen peptide was synthesized by the method of Geysen, cited above,for use in ELISA assays for detection of reactivity andcross-reactivity. Additional peptides with N- and C-terminal truncationswere also synthesized.

[0175] For most of the experiments reported below in Tables 2 and 3,detector peptides had an N-terminal -Ser-Gly-Ser-Gly- (SEQ ID NO: 30)added, with biotinylation of the new N-terminus, and the C-terminalremained a free acid. The C-terminal detecting peptides for some ofthese experiments had been inadvertently synthesized with an amidatedC-terminus, which may have led to a spuriously high binding for (SEQ IDNO: 32) -Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser- reported inTables 2 and 3 below. Therefore, several peptides were resynthesizedwith appropriate C-terminal groups and the pertinent parts of theexperiment were repeated with antibody to-Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser- (SEQ ID NO:28),and the results reported in Table 3 below. These detector peptides had apurity exceeding 70% by mass spectometry and were not purified further.

[0176] C. Immunization of Rabbits

[0177] The peptide conjugates were taken up in purified water andemulsified 1:1 with complete Freund's adjuvant (CFA) or incompleteFreund's adjuvant (IFA) (ANTIBODIES—A LABORATORY MANUAL, Eds. E. Harlowand P Lane, Cold Spring Harbor Laboratory (1998)). Total volume perrabbit was 1 ml, and this contained 100 μg of peptide coupled to DT.

[0178] Two rabbits were used for the immunizing peptide, with theinitial intramuscular (IM) injection with conjugate in CFA and asubsequent IM boost at 2 weeks with conjugate in IFA. A pre-bleed wasdrawn before the first injection and larger bleeds were taken 3 and 5weeks after the booster injection.

[0179] D. ELISA Determination of Binding of Antiserums to BiotinylatedPeptides

[0180] These assays were performed as described by H. M. Geysen et al.,Proc. Natl. Acad. Sci. USA, 81:3998 (1983). Briefly, using Nunc ImmunoMaxisorb™ 96 well plates, biotinylated peptides were bound tostreptavidin coated plates and, with washing with phosphate bufferedsaline (PBS) between steps, successive incubations were performed withantiserum dilutions and horseradish peroxidase conjugated anti-rabbitimmunoglobulin to detect bound antibody. Plates were developed withABTS, with an O D. reading at 405 nm. Absorbance greater than O D 1.0was taken as positive and titers were determined from doubling dilutionsof each antiserum. The geometric mean titer (GMT) was calculated foreach antiserum pair for a given immunogen. TABLE 2 Antiserum to EpitopeI: -Val- Asp-Pro-Arg-Leu-Glu-Pro-Trp- SEQ Lys-His-Pro-Gly-Ser-; GMT (%ID Detector Sequence* binding versus immunogen) NO.-Val-Asp-Pro-Arg-Leu-Glu- 65,885 (100) 31 Pro-Trp-Lys-His-Pro-Gly- Ser--Asp-Pro-Arg-Leu-Glu-Pro- 83,753 (127) 32 Trp-Lys-His-Pro-Gly-Ser--Val-Asp-Pro-Arg-Leu-Glu- 96,627 (147) 33 Pro-Trp--Val-Asp-Pro-Arg-Leu-Glu- 80,960 (123) 34 Pro- -Val-Asp-Pro-Arg-Leu-Glu-32,016 (49)  35

[0181] TABLE 3 % SEQ N C bind- ID Terminus Detector Sequences Terminusing No N0 Val-Asp-Pro-Arg-Leu-Glu-Pro-  C + 11 124 71Trp-Lys-His-Pro-Gly-Ser-Gln- Pro-Lys-Thr-Ala-OH N0Val-Asp-Pro-Arg-Leu-Glu-Pro- C + 6 100 28 Trp-Lys-His-Pro-Gly-Ser-OH N0Val-Asp-Pro-Arg-Leu-Glu-Pro- C + 1 120 33 Trp-OH N0Val-Asp-Pro-Arg-Leu-Glu-Pro- C + 1 134 92 Trp-NH₂ N0Val-Asp-Pro-Arg-Leu-Glu-Pro- C0 116 93 NH₂ N0Val-Asp-Pro-Arg-Leu-Glu-NH₂ C − 1 47 94 N0 Val-Asp-Pro-Arg-Leu-OH C − 25 95 N − 1 Asp-Pro-Arg-Leu-Glu-Pro- C + 1 65 96 Trp-OH N − 2Gly-Pro-Arg-Leu-Glu-Pro- C + 1 1 97 Trp-OH N − 2Ala-Pro-Arg-Leu-Glu-Pro- C + 1 1 98 Trp-OH

[0182] ELISA results reported in Tables 2 and 3 demonstrated that theantibodies to the first immunogen were reacting with the sequence-Asp-Pro-Arg-Leu-Glu-Pro (AA 5-10 of SEQ ID NO: 1). N- or C-terminaltruncation of these sequences reduced the ELISA titer. From the resultsof Tables 2 and 3 taken together, the N-terminal Val of the Epitope Ipeptide makes a small contribution to antibody binding (deletion gives65% of binding). Thus the definition of the primary antibody bindingregion of HIV-1 Tat, referred to herein as Epitope I, is:-Val-Asp-Pro-Arg-Leu-Glu-Pro- (SEQ ID NO:34).

[0183] E. HIV-1 Tat Epitope I Minimal Sequence Analysis

[0184] The following experiment was performed using as the immunizingpeptide sequence:Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser-NH₂ (SEQ ID NO: 28).As demonstrated by the results in Table 3, the minimal sequence ofEpitope I with maximal binding of antibody is confirmed asVal-Asp-Pro-Arg-Leu-Glu-Pro (SEQ ID NO:34). The variations in bindingwith C-terminal extensions are not significant. The truncation ofN-terminal Val or C-terminal Pro lead to modest reduction of bindingtiters, but truncating beyond this leads to almost complete loss ofspecific binding, indicating that Asp-Pro-Arg-Leu-Glu (amino acids 5-9of SEQ ID NO: 1) are the most important amino acids to createinteractions with specific antibody (See, e.g., FIG. 3). GMT is reportedas % of GMT on detector peptide containing the immunogen sequence.

[0185] F. Effects of Epitope I Immunogen Extension on Antibody Titers

[0186] N terminal extension of the epitope sequence in the immunogendoes not affect immunogenicity. In contrast, C terminal extension up toVal-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser-NH₂ (SEQ ID NO: 28)results in a 10-fold enhancement on titer. The present optimalimmunizing sequence appears to be SEQ ID NO: 28. GMT on SEQ ID NO: 28 isreported as % of GMT of antiserums to SEQ ID NO: 28 on this peptide, asshown in Table 4. TABLE 4 % Seq N C bind- ID Terminus Immunogen SequenceTerminus ing # N0 Val-Asp-Pro-Arg-Leu-Glu-Pro- C + 6 100 28Trp-Lys-His-Pro-Gly-Ser-NH₂ N + 2 Glu-Pro-Val-Asp-Pro-Arg-Leu- C + 6 10599 Glu-Pro-Trp-Lys-His-Pro-Gly- Ser-OH N0 Val-Asp-Pro-Arg-Leu-Glu-Pro-C + 4 10 100 Trp-Lys-His-Pro-OH N0 Val-Asp-Pro-Arg-Leu-Glu-Pro- C + 2 67101 Trp-Lys-OH N0 Val-Asp-Pro-Arg-Leu-Glu-Pro- C0 9 102 NH₂

[0187] G. Binding Pattern of Antibodies Induced by N-terminally ExtendedImmunogens

[0188] The binding of antiserums to Epitope I immunogens with N-terminalsequence extensions through the N-terminal Met were examined on detectorpeptides with full length and N- or C-terminal truncation. In theseinstances some of the immunizing peptides were synthesized withC-terminal Cys-amide for coupling to the carrier protein andcorresponding detector peptides were synthesized with aC-terminal-Gly-Ser-Gly-Ser-biocytin-amide (SEQ ID NO: 91) for binding toavidin coated plates. TABLE 5 Antiserum toMet-Glu-Pro-Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser-Gln-Pro-Lys-Thr-Ala- (amino acids 1-21 of SEQ ID NO:1). Titers on N-terminal truncation peptides % titer on longest DetectorPeptide Titer peptide Met-Glu-Pro-Val-Asp-Pro-Arg-Leu-Glu- 61,000 100Pro (amino acids 1-10 of SEQ ID NO: 1) -Glu-Pro-Val-Asp-Pro-Arg-Leu-Glu-54,000 89 Pro (amino acids 2-10 of SEQ ID NO: 1)-Pro-Val-Asp-Pro-Arg-Leu-Glu- 37,000 61 Pro (amino acids 3-10 of SEQ IDNO: 1) -Val-Asp-Pro-Arg-Leu-Glu- 19,000 22 Pro (amino acids 4-10 of SEQID NO: 1) -Pro-Arg-Leu-Glu- 9,000 11 Pro (amino acids 6-10 of SEQ IDNO: 1) -Arg-Leu-Glu-Pro 581 <1 (amino acids 7-10 of SEQ ID NO: 1)

[0189] TABLE 6 Antiserum to Glu-Pro-Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser (amino acids 2-16 of SEQ ID NO: 1). Titers onC-terminal truncation peptides % titer on longest Detector Peptide Titerpeptide Met-Glu-Pro-Val-Asp-Pro-Arg-Leu-Glu- 112,000 100 Pro (aminoacids 1-10 of SEQ ID NO: 1) Met-Glu-Pro-Val-Asp-Pro-Arg-Leu-Glu 27,00024 (amino acids 1-9 of SEQ ID NO: 1) Met-Glu-Pro-Arg-Leu 6,000 5 (aminoacids 1-5 of SEQ ID NO: 1)

[0190] From the above data it is evident that N-terminal extension ofthe immunizing Epitope I peptide extends the antibody binding regionthrough Glu₂ of the HIV-1 Tat protein sequence. TABLE 7 Titer on:Met-Glu- Titer on: Pro-Val-Asp-Pro- Val-Asp-Pro-Arg- Arg-Leu-Glu-ProLeu-Glu-Pro (amino acids 1-10 (amino acids 4-10 Immunizing Peptide ofSEQ ID NO: 1) of SEQ ID NO. 1) Val-Asp-Pro-Arg-Leu- 79,000 79,000Glu-Pro-Trp-Lys-His- Pro-Gly-Ser (amino acids 4-16 of SEQ ID NO: 1)Glu-Pro-Val-Asp-Pro- 70,000 42,000 Arg-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser (amino acids 2-16 of SEQ ID NO: 1)Met-Glu-Pro-Val-Asp- 61,000 19,000 Pro-Arg-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly- Ser-Gln-Pro-Lys-Thr- Ala-(amino acids 1-21 of SEQID NO: 1)

[0191] These data show that very similar total anti-Tat antibody titersare obtained with the various N-terminal immunogens but with a slightlydifferent distribution of the binding regions.

EXAMPLE 2 Sequence Variations in Epitope I of HIV-1 Tat Protein andImmunological Cross-reactivities of Antiserums to these Sequences

[0192] Variations in the sequence of Tat protein AA 5-10 of SEQ ID NO:1were analyzed in sequences available in HUMAN RETROVIRUSES and AIDS1996, published by the Theoretical Biology and Biophysics Group of theLos Alamos National Laboratory, Los Alamos, N. Mex., and additionalsequences kindly obtained from GenBank by Esther Guzman of the LosAlamos Laboratory.

[0193] A. Variations in Sequences

[0194] 399 aa 5-10 Tat hexapeptide sequences of the common B subtype ofHIV-1 were obtained, as were 18 from the non-B subtypes (6 from subtypeA, 2 from subtype C, 7 from subtype D, 2 from subtype F and 1 fromsubtype U).

[0195] For the B subtype, 386 of the total 399 (97%) hexapeptides hadeither Arg (289, 74%), or Lys (45, 11%), or Ser (36, 9%) or Asn (16, 4%)in position 3 as the only variation in the hexapeptides The remainingvariations (3%) comprised: The remaining variations (3%) comprised:-Gly-Pro-Arg-Leu-Glu-Pro-(4), (SEQ ID NO: 11)-Asp-Pro-Gly-Leu-Glu-Pro-(2), (SEQ ID NO: 14) and single examples of:-Asp-His-Arg-Leu-Glu-Pro-, (SEQ ID NO: 41) -Ala-Pro-Arg-Leu-Glu-Pro-,(SEQ ID NO: 12) -His-Pro-Arg-Leu-Glu-Pro-, (SEQ ID NO: 13)-Asp-Pro-Arg-Ile-Glu-Pro-, (SEQ ID NO: 15) -Asp-Pro-Arg-Leu-Gly-Pro-,(SEQ ID NO: 16) -Asp-Pro-Arg-Leu-Glu-Ala-and (SEQ ID NO: 17)-Asn-Pro-Ser-Leu-Glu-Pro-. (SEQ ID NO: 18)

[0196] For the 18 non-B subtype sequences, 2 had Arg, 1 had Lys, 2 hadSer and 9 had Asn at position 3 of the hexapeptides aa5-10, and othervariants were For the 18 non-B subtype sequences, 2 had Arg, 1 had Lys,2 had Ser and 9 had Asn at position 3 of the hexapeptides aa5-10, andother variants were -Asp-Pro-Asn-Leu-Asp-Pro- (2) (SEQ ID NO: 42) andsingle examples of -Asp-Pro-Asn-Ile-Glu-Pro- and (SEQ ID NO: 43)-Asp-Pro-Asn-Leu-Glu-Ser-. (SEQ ID NO: 44)

[0197] B. Assessment of Immunological Reactivity and Cross-reactivity ofthe Four Primary Immunogens

[0198] Immunizing and detector sequences were synthesized, as describedin Example 1, for the following sequences (SEQ ID NOS: 28 and 45 through47, respectively):

[0199] -Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser-,

[0200] -Val-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser-,

[0201] -Val-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser-,

[0202] -Val-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser-.

[0203] Rabbits were immunized and the antiserums were tested by ELISA,as described in Example 1, for reactivity and cross-reactivity.Self-reactivities are summarized in Table 8. TABLE 8 Immunogen anddetector sequence GMT SEQ ID NO-Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-His- 88,000 28 Pro-Gly-Ser--Val-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Lys-His- 132,000 45 Pro-Gly-Ser--Val-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Lys-His- 166,355 46 Pro-Gly-Ser--Val-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-His- 173,097 47 Pro-Gly-Ser-

[0204] Cross-reactivities between these primary immunogens with varyingamino acid residues at position 3 of Epitope I are displayed in Table 9.Note that the results reported below are averages with one poorlyreactive antiserum. TABLE 9 Antiserums to primary immunogens (figuresdenote % reactivity v. self-reactivity) Detectors Arg3 Lys3 Ser3 Asn3Arg3 100 49 3 4 Lys3 24 100 6 5 Ser3 11 16 100 15 Asn3 11 22 10 100

[0205] Tables 8 and 9 demonstrate that each variant is an effectiveimmunogen, but in general there is only modest cross-reactivity betweenvariants. The best cross-reactivity is obtained with the Lys3-containing immunogen. This implies that optimal coverage would requireinclusion of all four variants as immunogens in a primary composition asdescribed above.

[0206] C. Assessment of Cross-reactivities of Other Variants.

[0207] Detector peptides were made for all remaining epitope variantsand tested for cross-reactivity with the antiserums to the appropriateposition 3 primary hexapeptide immunogen. The cross-reactivities versusself-reactivity with the appropriate position 3 primary immunogen aredisplayed in Table 10. TABLE 10 Detector Sequence (variations in %Cross- SEQ ID Reactivity Epitope I) reactivity NO: Cross--Asp-His-Arg-Leu-Glu-Pro- 55 41 reactive ″ -Asp-Pro-Asn-Ile-Glu-Pro- 7043 ″ -Asp-Pro-Asn-Leu-Asp-Pro- 100 42 ″ -Asp-Pro-Asn-Leu-Glu-Ser- 78 44Non-cross -Gly-Pro-Arg-Leu-Glu-Pro- 1 11 reactive ″-Ala-Pro-Arg-Leu-Glu-Pro- 1 12 ″ -His-Pro-Arg-Leu-Glu-Pro- 1 13 ″-Asp-Pro-Gly-Leu-Glu-Pro- 1 14 ″ -Asp-Pro-Arg-Ile-Glu-Pro- 9 15 ″-Asp-Pro-Arg-Leu-Gly-Pro- 10 16 ″ -Asp-Pro-Arg-Leu-Glu-Ala- 1 17 ″-Asn-Pro-Ser-Leu-Glu-Pro- 10 18

[0208] The results of Tables 8-10 indicate that immunization with thefour primary immunogens would generate antibodies reactive with greaterthan 97% of HIV-1 Tat proteins of the common B subtype. Interestinglyall 18 non-B subtypes in the databases had Epitope I sequences reactivewith antibodies to the primary immunogens.

[0209] D. Immunogenicity of Certain Epitope I Variants

[0210] Immunizing and detecting peptides were synthesized for thefollowing 2 variant Epitope I peptides, with immunizations and ELISAtesting as in Example 1. The self titers (GMT) are displayed in Table11. TABLE 11 Detecting and Immunizing Peptides SEQ ID (GMT) Self-titerNOS. -Val-Asn-Pro-Ser-Leu-Glu-Pro-Trp-Lys-His- 94,919 48 Pro-Gly-Ser--Val-Asp-His-Arg-Leu-Glu-Pro-Trp-Lys-His- 72,686 49 Pro-Gly-Ser-

[0211] These data show that inclusion of rare variants along with theprimary immunogens expands antibody coverage to such rare epitopevariants.

[0212] Immunization with the four primary Epitope I sequences can inducehigh titer antibodies reactive with Tat proteins of >97% of all HIV-1strains. This coverage can be optionally extended with the inclusion ofadditional rare Epitope I variant sequences in the immunizingcomposition.

[0213] E. Consequences of N-terminal Immunogen Extension onImmunological Conservation and Cross-Reactivity

[0214] Reviewing the Epitope I sequences studied above, the majorvariation in the Glu-Pro- extension is the occurrence of an Asp for Glusubstitution in 9% of sequences. However, as shown in Table 12,antibodies to Glu containing peptides are substantially cross-reactivewith the corresponding Asp containing peptide, and vice-versa. TABLE 12Detector Peptides (Titer) Met-Glu-Pro- Val-Asp- Val-Asp-Pro-Met-Asp-Pro- Pro-Arg- Arg-Leu-Glu- Val-Asp-Pro- Leu-Glu-Pro Pro (aminoArg-Leu-Glu- (amino acids acids 1-10 of Pro (SEQ ID 4-10 of ImmunogensSEQ ID NO: 1) NO: 103) SEQ ID NO: 1) Met-Glu-Pro-Val- 82,000 67,00038,000 Asp-Pro-Arg-Leu- Glu-Pro-Trp-Lys (amino acids 1-12 of SEQ IDNO: 1) Met-Asp-Pro-Val- 50,000 75,000 38,000 Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys (SEQ ID NO. 104)

[0215] Other than the variation between Glu and Asp there were only 7other variants at these two positions (5 Lys/Glu and 2 Leu-Prosubstitutions), bringing the immunological conservation of the enlargedEpitope I in B subtype HIV-1 Tat proteins to 95%. For the 18 non Bsubtype sequences only 16 contained sequence corresponding to theGlu-Pro extension and, apart from Glu/Asp variation, all were Glu-Pro orAsp-Pro except for two F subtype sequences (Glu-Leu), yielding animmunological conservation of 88%. Thus immunization with peptides withextended N-terminal sequence still provides a high incidence ofimmunoreactivity with a broad sample of known HIV-1 Tat proteinsequences.

EXAMPLE 3 Defining an Antibody Binding Amino Acid Sequence (Epitope II)within the Linear 18 Amino Acid Sequence Following Cys₃₇ of HIV-1 TatProtein

[0216] A peptide corresponding to amino acids 38-55 of SEQ ID NO: 1illustrated in FIG. 1 was synthesized as described in Example 1. Usingthe methods described in Example 1, a low titer antibody response inrabbits was detected and Table 28 summarizes studies defining thesequence involved in this antibody binding. The geometric mean titer(GMT) is reported as percentage of self-titer. TABLE 13 Antiserum to SEQID NO: 105: Phe-Ile-Thr-Lys-Gly-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg GMT (% Detector Peptides selftiter) SEQ ID NO. Phe-Ile-Thr-Lys-Gly-Leu-  947 (100) 105 Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys-Arg- Arg-Gln-Arg Phe-Ile-Thr-Lys-Gly-Leu- 1141 (115)(amino acids 1-15 of Gly-Ile-Ser- SEQ ID NO: 105)Tyr-Gly-Arg-Lys-Lys-Arg Lys-Gly-Leu-Gly-Ile-Ser- 895 (95) (amino acids41-55 of Tyr-Gly-Arg-Lys-Lys-Arg- SEQ ID NO: 1) Arg-Gln-ArgLeu-Gly-Ile-Ser-  986 (104) (amino acids 43-55 ofTyr-Gly-Arg-Lys-Lys-Arg- SEQ ID NO: 1) Arg-Gln-Arg Gly-Ile-Ser- 428 (47)(amino acids 44-52 of Tyr-Gly-Arg-Lys-Lys-Arg SEQ ID NO: 1) Ile-Ser- 254(27) (amino acids 45-52 of Tyr-Gly-Arg-Lys-Lys-Arg SEQ ID NO: 1) Ser- 53(6) (amino acids 46-55 of Tyr-Gly-Arg-Lys-Lys-Arg- SEQ ID NO: 1)Arg-Gln-Arg Leu-Gly-Ile-Ser-Tyr-Gly- 914 (97) (amino acids 43-51 ofArg-Lys-Lys SEQ ID NO: 1) Leu-Gly-Ile-Ser-Tyr-Gly- 545 (58) (amino acids43-50 of Arg-Lys SEQ ID NO: 1) Phe-Ile-Thr-Lys-Gly- 129 (14) (aminoacids 1-12 of Leu-Gly-Ile-Ser-Tyr-Gly- SEQ ID NO: 105) Arg

[0217] These ELISA results established that the low titer antibodiesinduced bound to the sequence Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys (aminoacids 43-51 of SEQ ID NO: 1). Accordingly, this sequence and thesequence Lys-Gly-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys (amino acids 41-51of SEQ ID NO: 1) were synthesized and used to immunize rabbits.

[0218] Immunizing with the minimal epitope,Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys (amino acids 43-51 of SEQ ID NO: 1),produced low titer antibodies (GMT 1,000) while, surprisingly,immunization with Lys-Gly-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys (aminoacids 41-51 of SEQ ID NO: 1) induced high titer antibodies (GMT 20,000)to the detector peptidePhe-Ile-Thr-Lys-Gly-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg(SEQ ID NO: 105).

[0219] The variation of sequence occurring within Epitope II wasanalyzed to enable the design of immunogens that induce antibodies thatreact with most Epitope II sequences. 441 sequence variations weredetermined in HIV-1 Tat protein amino acids 41-51 of the common Bsubtype were obtained, as were 21 from non-B subtypes (7 from subtype A,4 from subtype C, 7 from subtype D, 2 from subtype F and 1 from subtypeU). For the B subtype, 422 of 441 (96%) had the nominal sequence withthe exception of an Ala for Gly substitution in 134 (32%). For the non-Bsubtype sequences 20 of 21 (95%) had the nominal sequence.

[0220] The reactivity of antibodies induced byLys-Gly-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys (amino acids 41-51 of SEQ IDNO: 1) was studied by titering antiserums toLys-Gly-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys (amino acids 41-51 of SEQ IDNO: 1) as follows on the listed detector peptides: TABLE 14 DetectorPeptides Titer SEQ ID NO. Phe-Ile-Thr-Lys-Gly-Leu-Gly- 20,000 105Ile-Ser-Tyr-Gly-Arg-Lys-Lys- Arg-Arg-Gln-ArgLys-Gly-Leu-Gly-Ile-Ser-Tyr- 18,000 (amino acids 4-18 ofGly-Arg-Lys-Lys-Arg-Arg-Gln- SEQ ID NO: 105) ArgLys-Ala-Leu-Gly-Ile-Ser-Tyr- 20,000 106 Gly-Arg-Lys-LysLeu-Gly-Ile-Ser-Tyr-Gly- 19,000 (amino acids 3-11 of Arg-Lys-Lys SEQ IDNO: 106)

[0221] These data show that there is full immunological cross reactivitywith the Ala variant and that the antibody response toLys-Gly-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys (amino acids 41-51 of SEQ IDNO: 1) remains directed to the previously identified epitopeLeu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys (amino acids 43-51 of SEQ ID NO: 1).Thus immunization with Lys-Gly-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys(amino acids 41-51 of SEQ ID NO: 1) induces high titer antibodies whichreact with greater than 96% of known HIV-1 Tat proteins.

EXAMPLE 4 Immunological Studies on Minimal Tat Protein Amino AcidSequences Necessary for Binding to Antibody for Epitope III in HIV-1 TatProtein

[0222] A peptide was synthesized which corresponded to amino acids 53-62of SEQ ID NO: 1 illustrated in FIG. 1 as described below. The sequenceis the most frequent sequence representations at these positions in 31Tat protein sequences of the common B subtype reported in the NIAID HIVdatabase. The sequence was chosen as a second putative immunogen.

[0223] A. Peptide Synthesis—Immunizing Peptides

[0224] The amino acid sequences of the immunogen,-Arg-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- (SEQ ID NO: 29) wassynthesized as described in Example 1 for Epitope I. The experiments forwhich data is reported are represented in Tables 15-16 below. Immunizingpeptides were generally purified to greater than 95% purity by reversephase HPLC, and purity was further confirmed by mass spectometry (MS).Immunizing peptides were covalently coupled to diphtheria toxoid (DT)carrier protein via the cysteinyl side chain as described for Epitope Iin Example 1A.

[0225] B. Peptide Synthesis—Detector Peptides

[0226] Peptides corresponding to the amino acid sequence of theimmunogen peptide were synthesized by the method of Geysen, cited above,for use in ELISA assays for detection of reactivity andcross-reactivity. Additional peptides with N- and C-terminal truncationswere also synthesized. For most of the experiments reported below inTable 15, detector peptides had an N-terminal -Ser-Gly-Ser-Gly- (SEQ IDNO: 30) added, with biotinylation of the new N-terminus, and theC-terminal remained a free acid. These detector peptides had a purityexceeding 70% by mass spectometry and were not purified further.

[0227] C. Immunization of Rabbits

[0228] The peptide conjugates were taken up in purified water andemulsified 1:1 with CFA or IFA as described in Example 1C. Total volumeper rabbit was 1 ml, and this contained 100 μg of peptide coupled to DT.Two rabbits were used for the immunizing peptide, with the initialintramuscular (IM) injection with conjugate in CFA and a subsequent IMboost at 2 weeks with conjugate in IFA. A pre-bleed was drawn before thefirst injection and larger bleeds were taken 3 and 5 weeks after thebooster injection.

[0229] D. ELISA Determination of Binding of Antiserums to BiotinylatedPeptides

[0230] These assays were performed as described by H. M Geysen et al.,Proc. Natl Acad. Sci. USA, 81:3998 (1983) and as described in Example 1Dfor Epitope I. From the results of Table 15, the definition of anotherantibody binding region of HIV-1 Tat, referred to herein as Epitope III,is -Arg-Arg-Ala-Pro-Gln-Asp-Ser- (SEQ ID NO: 19).

[0231] E. HIV-1 Tat Epitope III Minimal Sequence Analysis

[0232] The following experiment was performed using as the immunizingpeptide sequence: Arg-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser-NH₂ (SEQ IDNO:29). The results in the following Table 15 illustrate that theminimal sequence of Epitope III with maximal binding of antibody isArg-Arg-Ala-Pro-Gln-Asp-Ser (SEQ ID NO: 19). N-terminal or C-terminalextensions of the detector peptides do not produce significantlydifferent binding. The truncation of N-terminal Arg leads to a modestreduction of binding titer, while truncation of the next N-terminal Argor the C-terminal Ser lead to almost complete loss of specific binding,indicating that Arg-Ala-Pro-Gln-Asp-Ser (amino acids 57-62 of SEQ IDNO: 1) are the most important amino acids to create interactions withspecific antibody (See FIG. 4). GMT is reported as % GMT on detectorpeptide containing the immunogen sequence. TABLE 15 N Detector C % TermSequences Term binding SEQ ID # N + 3 Arg-Gln-Arg-Arg-Arg-Ala- C0 100(amino acids 53-62 Pro-Gln-Asp-Ser-OH of SEQ ID NO: 1) N + 2Gln-Arg-Arg-Arg-Ala-Pro- C0 94 (amino acids 54-62 Gln-Asp-Ser-OH of SEQID NO: 1) N + 1 Arg-Arg-Arg-Ala-Pro-Gln- C0 93 (amino acids 55-62Asp-Ser-OH of SEQ ID NO: 1) N0 Arg-Arg-Ala-Pro-Gln- C0 105 (amino acids56-62 Asp-Ser-OH of SEQ ID NO: 1) N0 Arg-Arg-Ala-Pro-Gln- C + 4 107(amino acids 56-66 Asp-Ser-Gln-Thr-His-Gln- of SEQ ID NO: 1) OH N − 1Arg-Ala-Pro-Gln- C0 56 (amino acids 57-62 Asp-Ser-OH of SEQ ID NO: 1) N− 2 Ala-Pro-Gln- C0 3 (amino acids 58-62 Asp-Ser-OH of SEQ ID NO: 1) N +3 Arg-Gln-Arg-Arg-Arg-Ala- C − 1 7 (amino acids 53-61 Pro-Gln-Asp-OH ofSEQ ID NO: 1) N + 3 Arg-Gln-Arg-Arg-Arg-Ala- C − 2 4 (amino acids 53-60Pro-Gln-OH of SEQ ID NO: 1)

[0233] F. HIV-1 Tat Epitope III—Effects of Immunogen on Antibody Titers

[0234] Every instance of C-terminal extension results in loss ofimmunogenicity and lowered titers. In contrast, N terminal extension upto a point enhances immunogenicity, with maximal titers being obtainedwith Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser (amino acids 54-62 of SEQ IDNO: 1) and a drop in immunogenicity occurring withArg-Arg-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- (amino acids 52-62 of SEQID NO: 1) as the immunogen. GMT onArg-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- (amino acids 53-62 of SEQ IDNO: 1) is reported in Table 16 as % of GMT of antiserums to this peptideon this detector peptide. TABLE 16 N C % Term Immunogen Sequence Termbinding SEQ ID # N + 3 Arg-Gln-Arg-Arg-Arg-Ala- C0 100 107Pro-Gln-Asp-Ser-NH₂ N + 1 Gly-Arg-Arg-Ala-Pro- C0 11 108 Gln-Asp-Ser-NH₂N0 Arg-Arg-Ala-Pro- C + 4 3 109 Gln-Asp-Ser-Gln-Thr-His- Gln-NH₂ N + 2Gln-Arg-Arg-Arg-Ala-Pro- C0 136 (amino acids 54-62 Gln-Asp-Ser-OH of SEQID NO: 1) N + 3 Arg-Gln-Arg-Arg-Arg-Ala- C + 2 9 (amino acids 53-64Pro-Gln-Asp-Ser-Gln-Thr- of SEQ ID NO: 1) OH N + 3Arg-Gln-Arg-Arg-Arg-Ala- C + 4 28 (amino acids 53-66Pro-Gln-Asp-Ser-Gln-Thr- of SEQ ID NO: 1) His-Gln-OH N + 4Arg-Arg-Gln-Arg-Arg-Arg- C0 3 (amino acids 52-62 Ala-Pro-Gln-Asp-Ser-OHof SEQ ID NO: 1)

EXAMPLE 5 Sequence Variations in Epitope III of HIV-1 Tat Protein andImmunological Cross-reactivities of Antiserums to these Sequences

[0235] Variations in the sequence of Tat protein AA 56-72 were analyzedin sequences available in HUMAN RETROVIRUSES and AIDS 1996, (citedabove) and additional sequences (GenBank), as described in Example 4.

[0236] A. Variations in Epitope III Sequences

[0237] 482 sequences of Epitope III of the common B subtype of HIV-1were available for analysis. The most frequent sequence found conformedto the formula -Arg-Arg-X-Pro-Gln-Y-Ser- (SEQ ID NO: 110), where X isAla, Pro, Ser, or Gln, and Y is Asp, Asn, Gly or Ser. This sequencetype, which appears immunologically cross-reactive (see below), wasfound in 292 of 482 (61%) of available Tat sequences.

[0238] Other sequence variants occurred in lower incidence and theseincluded those listed in Table 17 below. TABLE 17 # (%) of Tat SEQUENCESSequences SEQ ID NO. -Arg-Arg-Ala-Pro-Pro-Asp-Ser- 20 (4%) 20-Arg-Arg-Ala-Pro-Pro-Asp-Asn- 21 (4%) 50 -Arg-Arg-Ala-His-Gln-Asp-Ser-20 (4%) 21 -Arg-Arg-Ala-His-Gln-Asn-Ser-   17 (3.5%) 22-Arg-Arg-Ala-Pro-Gln-Gly-Asn- 10 (2%) 51 -Arg-Gly-Ala-Pro-Gln-Asp-Ser- 9 (2%) 25 -Arg-Arg-Ala-Pro-Glu-Asp-Ser-  8 (2%) 26-Arg-Arg-Ala-Ser-Gln-Asp-Ser-  8 (2%) 27 -Arg-Arg-Pro-Pro-Gln-Asp-Asn- 9 (2%) 23 -Arg-Arg-Ala-Pro-Gln-Asp-Arg-  8 (2%) 24

[0239] Together these sequences account for 85% of Epitope III variants,with the balance comprised of a large number of low incidencevariations.

[0240] Epitope III sequences were only available from 18 examples ofHIV-1 non-B subtypes. Unlike Epitope I, they showed divergence from theB subtype sequences and optionally a larger number of sequences can beselected for inclusion into the composition of this invention, ifadditional non-B subtype Epitope III sequences are determined and aredesirable in an immunogenic composition of this invention.

[0241] B. Assessment of Immunological Reactivity and Cross Reactivity ofSelected Epitope III Sequences

[0242] Immunizing and detector sequences were synthesized, as describedin Example 1, for the sequences in Table 18 Rabbits were immunized andantiserums were tested by ELISA, as described in Example 4, forreactivity and cross-reactivity. The various antiserums and detectorpeptides were utilized to determine immunogenicity of the varioussequences and the extent of immunological cross reactivity. Theincidence and immunological reactivity of Epitope III sequences of theformula -Arg-Arg-X-Pro-Gln-Y-Ser- (SEQ ID NO: 10) (see above) are shownin Table 18. In Table 18 below, percent cross-reactivity was measuredwith antiserum to Cys-Arg-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- (SEQ IDNO: 74), self titer=46, 115. The results of Table 18 demonstrate thatimmunization with -Arg-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- (SEQ ID NO:29) should provide effective cross-reactivity with most of thesevariants, represented in 61% of HIV-1 strains. TABLE 18 # per 482 %Cross- SEQ ID Epitope III Sequence sequences reactivity NO.-Arg-Arg-Ala-Pro-Gln-Asp-Ser- 93 100 19 -Arg-Arg-Pro-Pro-Gln-Asp-Ser- 50111 75 -Arg-Arg-Pro-Pro-Gln-Asn-Ser- 41 96 76-Arg-Arg-Pro-Pro-Gln-Gly-Ser- 37 97 77 -Arg-Arg-Ser-Pro-Gln-Asp-Ser- 1993 73 -Arg-Arg-Thr-Pro-Gln-Gly-Ser- 14 56 68-Arg-Arg-Ala-Pro-Gln-Gly-Ser- 9 87 69 -Arg-Arg-Thr-Pro-Gln-Asp-Ser- 7116 70 -Arg-Arg-Ala-Pro-Gln-Asn-Ser- 5 128 72-Arg-Arg-Ser-Pro-Gln-Asp-Ser- 4 110 73 -Arg-Arg-Ser-Pro-Gln-Asn-Ser- 2142 78 -Arg-Arg-Ala-Pro-Gln-Ser-Ser- 1 97 79-Arg-Arg-Ser-Pro-Gln-Gly-Ser- 1 78 80 -Arg-Arg-Thr-Pro-Gln-Asn-Ser- 1 4381 292/482 (61%)

[0243] Immunization with -Arg-Arg-Ala-Pro-Pro-Asp-Asn- (SEQ ID NO: 50)and -Arg-Arg-Ala-Pro-Pro-Asp-Ser- (SEQ ID NO: 20) yielded antibodiesthat cross-reacted with both detector peptides, as shown in Table 19.Thus, inclusion of either sequence in an immunizing composition of thisinvention provides antibodies against Tat protein Epitope III variantsin a further 41/482 (8.5%) of HIV-1 strains.

[0244] Immunization with -Arg-Gln-Arg-Arg-Arg-Ala-His-Gln-Asn-Ser- (SEQID NO: 52) (20/482 (4%)) induced antibodies that gave a self titer of209,286 and a cross-reactivity of 5,356 (2.5%) with-Arg-Arg-Ala-His-Gln-Asp-Ser- (SEQ ID NO: 21) (17/482 (3.5%)). Thusinclusion of this sequence in an immunogen covers an additional 27/482(7.5%) of HIV-1 strains.

[0245] Thus, immunization with three Epitope III variants,

[0246] -Arg-Arg-Ala-Pro-Gln-Asp-Ser- (SEQ ID NO: 19),

[0247] -Arg-Arg-Ala-Pro-Pro-Asp-Asn- (SEQ ID NO: 50), and

[0248] -Arg-Arg-Ala-His-Gln-Asn-Ser- (SEQ ID NO. 22), providesantibodies reactive with the Tat proteins of 77% of HIV-1 strains. TABLE19 Detector peptides ELISA GMT -Arg-Arg-Ala-Pro- -Arg-Arg-Ala-Pro-Antiserum Pro-Asp-Asn- Pro-Asp-Ser- to: (SEQ ID NO: 50) (SEQ ID NO: 20)SEQ ID NO: 50 11,056 12,230 SEQ ID NO: 20 9,340 7,865

EXAMPLE 6 Immunological Studies on Minimal Tat Protein Amino AcidSequences Necessary for Binding to Antibody for Epitope IV in HIV-1 TatProtein

[0249] A publication by McPhee at al, FEBS Letters 233:393 (1988)suggested that some serums of HIV-1 infected subjects reacted with asynthetic peptide Ser-Gln-Thr-His-Gln-Val-Ser-Leu-Ser-Lys-Gln-Pro-Cys(SEQ ID NO: 111), erroneously reported as amino acids 71-83 of HIV-1 Tatprotein (the correct positions are amino acids 61-73). The inventortherefore immunized mice with the syntheticpeptide-Ser-Gln-Thr-His-Gln-Val-Ser-Leu-Ser-Lys-Gln-Pro (SEQ ID NO: 112)and determined that antibodies reactive with this peptide weregenerated, with a geometric mean titer of 26,517.

[0250] To determine minimal epitope size, a series of truncated peptideswere synthesized and used as detector peptides to determine minimalsequence length requirements for binding. Table 20 reports the detectorpeptides and percent binding, determined as described in Example 1 forEpitope I. TABLE 20 % Detector Peptides Binding SEQ ID NO-Ser-Gln-Thr-His-Gln-Val-Ser-Leu- 100 112 Ser-Lys-Gln-Pro-(GMT 26,517)-Gln-Thr-His-Gln-Val-Ser-Leu-Ser- 63 (amino acids 2-12 of Lys-Gln-Pro-SEQ ID NO: 112) -Thr-His-Gln-Val-Ser-Leu-Ser- 42 (amino acids 3-12 ofLys-Gln-Pro- SEQ ID NO: 112) -His-Gln-Val-Ser-Leu-Ser- 16 (amino acids4-12 of Lys-Gln-Pro- SEQ ID NO: 112) -Gln-Val-Ser-Leu- 5 (amino acids5-12 of Ser-Lys-Gln-Pro- SEQ ID NO: 112)-Ser-Gln-Thr-His-Gln-Val-Ser-Leu- 15 (amino acids 1-11 of Ser-Lys-Gln-SEQ ID NO: 112) -Ser-Gln-Thr-His-Gln-Val-Ser-Leu- 3 (amino acids 1-10 ofSer-Lys- SEQ ID NO: 112)

[0251] From these data, it is clear that all 12 amino acids arenecessary for full binding to Epitope IV, although the contribution ofamino acids in positions 1 and 2 are not major.

[0252] The following determinations were made about sequence variationsin Epitope IV and immunological cross-reactivies of antiserums,following the procedures described above for Epitopes I, II and III. 444examples of this sequence region were available in the data bases. Themost common variations were Asn for Thr at position 3 and Ala for Val atposition 6. Reactivities of the antiserums to the nominal peptide todetector peptides corresponding to these sequences were studied asdescribed above, and the results of percent binding reported in Table 21below. TABLE 21 Detector Peptides % Binding SEQ ID NO.-Ser-Gln-Thr-His-Gln-Val-Ser-Leu-Ser-Lys- 100 112 Gln-Pro-(GMT 26,517)-Ser-Gln-Asn-His-Gln-Val-Ser-Leu-Ser-Lys- 85 113 Gln-Pro--Ser-Gln-Thr-His-Gln-Ala-Ser-Leu-Ser-Lys- 17 114 Gln-Pro-

[0253] Thus amino acid position 3 substitution from Thr to Asn does notmaterially affect antibody binding, whereas an Ala for Val substitutionat amino acid position 6 is essentially non cross-reactive. In the 444examples of HIV-1 Tat proteins sequenced in this region 282/444 (64%)had sequences that were similar to the nominal sequence. Disregardingamino acid 3 variations between Thr and Asn, 199/444 (45%) had Val asamino acid 6 and 83/444 (19%) had Ala as amino acid 6. In contrast tothe poor cross-reactivity of Ala6 peptides with antisera to the Val6immunogen, antisera to Ala6 peptide gave titers of 26,000 on the Ala6peptide, and 32,000 on the Val6 peptide, demonstrating completecross-reactivity.

[0254] Thus immunization with proteins containing the amino acidsequence -Ser-Gln-Thr-His-Gln-Ala-Ser-Leu-Ser-Lys-Gln-Pro- (SEQ ID NO:114) provokes antibodies reactive with 64% of variant HIV-1 Tatproteins.

EXAMPLE 7 Construction of a Synthetic Gene of the Invention

[0255] A synthetic gene was constructed that incorporated in frame eightEpitope I variants (including the four primary immunogens of theinvention) and thirteen Epitope III variants, these constituting all thevariant Epitope I and Epitope III sequences found in the Tat proteinsequences of 31 HIV-1 B subtype strains reported in the 1996 HUMANRETROVIRUSES and AIDS compilation, cited above. These included aminoacids 4-16 for Epitope I and 53-62 for Epitope III, using the numberingof SEQ ID NO: 1 illustrated in FIG. 1. The epitope sequences wereseparated by dipeptide spacers containing Gly and/or Ser residues.

[0256] The sequence of this one exemplary gene of this invention isshown in FIGS. 2A-2C (SEQ ID NOS: 2 and 3). The gene was assembled asdescribed in W. P. C. Stemmer et al., Gene, 164:49 (1995). Briefly,eleven top strand 60-mer oligonucleotides (oligos) and eleven bottomstrand oligos with 20 nucleotide (nt) overlaps were synthesized alongwith two end 50-mers. The twenty-two 60-mers were incubated togetherunder hybridizing conditions and polymerase chain reaction (PCR) wasused to fill in the sequence and amplify it. The end 50-mers were thenadded and the assembly completed by PCR, with isolation of the fulllength gene on agarose gel.

[0257] The gene was sequenced and found to have the correct sequencewithin the actual epitopes, with the exception of an Ala to Thrsubstitution at position 136 (see FIGS. 2A-2C) This was accepted sincethis change does not affect antibody binding of Epitope III (see Example4).

[0258] This gene was then excised with restriction enzymes and insertedinto the expression vector pBAD (L -M. Guzman et al., J. Bacteriol,177:4121 (1950)) containing, in frame, the sequence for greenfluorescent protein (GFP) (A. Crameri et al., Nature Biotech, 14:315(1996)). TG1 E. coli were transfected and green-fluorescent colonieswere isolated.

[0259] The isolated colonies were grown and expression was induced.Protein from each of three colonies had fluorescent bands on Westernblotting with the expected molecular size (i.e., twice that of GFPalone). The resulting protein was soluble and was purified by nickelcolumn affinity purification utilizing a hexa-histidyl that had beenincorporated in the sequence.

[0260] Yield was approximately 1 mg protein per liter of supernatantafter double affinity purification to yield >90% purity.

EXAMPLE 8 Immunological Characterization of the Recombinant FusionProtein Expressing HIV-1 Tat Protein Epitope Variants

[0261] A. Reactivity of Fusion Protein with Rabbit Antiserums to EpitopeI and 2 Variants.

[0262] Rabbit antiserums generated to synthetic peptides correspondingto the four primary Epitope I sequences and four Epitope III sequences(see below) were tested by ELISA, using the methodology described inExamples 1 and 4, except that the plates were initially directly coatedwith a 100 μg/ml solution of antiserums with the fusion protein. TABLE22 Titers on fusion SEQ ID Antiserum to: protein NOS-Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-His-Pro- >>8000 28 Gly-Ser--Val-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-His-Pro- >>8000 47 Gly-Ser--Val-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Lys-His-Pro- >>8000 45 Gly-Ser--Val-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Lys-His-Pro- >8000 46 Gly-Ser--Arg-Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Ser- 7000 29-Arg-Gln-Arg-Arg-Arg-Ala-His-Gln-Asn-Ser- >>8000 52-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Asp-Ser- >8000 53-Arg-Gln-Arg-Gln-Arg-Ala-Pro-Asp-Ser-Ser- 8000 82

[0263] These data show that the variant epitope sequences, expressed asa linear recombinant fusion protein, are expressed in a conformationrecognizable by antibodies to the corresponding synthetic peptides.

[0264] B. Immunization of Mice with the Fusion Protein

[0265] Three mice were immunized with 10 μg each of an aqueous solutionof the fusion protein of Example 7 emulsified with an equal volume ofFreund's complete adjuvant, given intraperitoneally Two weeks later theywere similarly boosted, except that Freund's incomplete adjuvant wasused. Serums were obtained three weeks later.

[0266] C. ELISA Testing of Antiserums to the Fusion Protein withSynthetic Peptides Corresponding to the Epitope Variants Incorporated inthe Fusion Protein

[0267] ELISA testing was performed as described in Example 1 except thathorseradish peroxidase conjugated anti-mouse immunoglobulin was used todetect antibody binding. The results are summarized in Table 23 below.These data demonstrate that both Epitope I and Epitope III sequences areexpressed in the linear fusion protein, and react with antibodies to thesynthetic sequences (see above). Antibodies to Epitope I were detectablyinduced by the recombinant fusion protein under the conditions of thisexperiment in mice. The failure of Epitope III sequences expressedwithin this linear peptide is in keeping with the findings withsynthetic peptides, wherein C-terminal extension of the immunogendiminishes the resulting antibody titer (see Example 4). TABLE 23 Titerwith antiserum to fusion Detector peptides protein SEQ ID NO-Val-Asp-Pro-Arg-Leu-Glu-Pro-Trp- 2218 28 Lys-His-Pro-Gly-Ser-Val-Asp-Pro-Asn-Leu-Glu-Pro-Trp- 3158 47 Lys-His-Pro-Gly-Ser--Val-Asp-Pro-Lys-Leu-Glu-Pro-Trp- 2440 45 Lys-His-Pro-Gly-Ser--Val-Asp-Pro-Ser-Leu-Glu-Pro-Trp- 3031 46 Lys-His-Pro-Gly-Ser--Val-Asn-Pro-Ser-Leu-Glu-Pro-Trp- 3718 48 Lys-His-Pro-Gly-Ser--Val-Asp-His-Arg-Leu-Glu-Pro-Trp- 3223 49 Lys-His-Pro-Gly-Ser--Arg-Gln-Arg-Arg-Arg-Ala-Pro-Gln- background 29 Asp-Ser--Arg-Gln-Arg-Arg-Arg-Ala-His-Gln- background 52 Asn-Ser--Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln- background 53 Asp-Ser--Arg-Gln-Arg-Gln-Arg-Ala-Pro-Asp- background 82 Ser-Ser--Arg-Gln-Arg-Arg-Arg-Ala-Pro-Glu- background 83 Asp-Ser--Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln- background 59 Gly-Ser--Arg-Gln-Arg-Arg-Gly-Pro-Pro-Gln- background 60 Gly-Ser--Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln- background 61 Asn-Ser--Arg-Gln-Arg-Arg-Arg-Ser-Pro-Gln- background 62 Asp-Ser--Arg-Gln-Arg-Arg-Arg-Ser-Pro-Gln- background 63 Asn-Ser--Arg-Gln-Arg-Arg-Arg-Thr-Pro-Gln- background 64 Ser-Ser--Arg-Gln-Arg-Arg-Arg-Ala-His-Gln- background 65 Asp-Ser--Arg-Gln-Arg-Arg-Arg-Ala-Pro-Pro- background 66 Asp-Ser-

EXAMPLE 9 Primate Animal Study

[0268] A study was conducted in ten juvenile male rhesus macaques todetermine if the presence of antibodies to Tat protein, induced by asynthetic peptide of this invention prior to infection withimmunodeficiency virus, would attenuate infection and reduce levels ofvirus in plasma. HIV-1 does not infect monkeys, but a correspondingsimian immunodeficiency virus (SIV) does. P A. Luciw et al., Proc. NatlAcad. Sci USA, 92:7490 (1995) constructed an infectious recombinantvirus (chimera) of SIV_(mac239) and HIV-1_(SF33) that does infectmonkeys, typically causing an acute viremia that peaks around 2 weeksand subsequently subsides by week 8. In this chimeric construct, termedSHIV_(SF33), the SIV nucleotides encoding tat, rev and env (gp160) ofSIV_(mac239) have been replaced with the corresponding region ofHIV-1_(SF) ₃₃.

[0269] A. Immunization of Monkeys

[0270] The monkeys were randomized into two groups.

[0271] Each monkey of group 1 (control group) was immunized with 0.4 mgdiphtheria toxoid (Commonwealth Scrum Laboratories, Victoria, Australia)with 0.25 mg threonyl muramyl dipeptide (T-MDP) in 0.5 ml water, thisbeing emulsified with 0.5 ml MF75 adjuvant (Chiron Corp, EmeryvilleCalif.).

[0272] Each monkey of group 2 (test group) was immunized with 0.1 mg ofthe synthetic peptideCys-Val-Asp-Pro-Asn-Leu-Glu-Pro-Trp-His-Pro-Gly-Ser- amide (SEQ ID NO:84) coupled to 0.4 mg diphtheria toxoid (A. C. Lee et al., Mol.Immunol., 17:749 (1980)). The conjugate was dissolved in 0.5 ml watercontaining 0.25 mg T-MDP and emulsified with 0.5 ml MF75 adjuvant.

[0273] Each monkey was immunized at day 0 and day 28 (week 4) with two0.5 ml intramuscular injections at two distinct sites. The syntheticpeptide immunogen contained the B cell Epitope I,Val-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser- (SEQ ID NO: 115) ofthe Tat protein of SF33 HIV-1 that is incorporated in the SHIV_(SF33)molecular clone that was used to challenge the monkeys (see above).

[0274] B. Testing for Antibodies to Tat Protein

[0275] At day 42 (week 6), 2 weeks after the booster injection, serumswere drawn and tested by ELISA for binding toSer-Gly-Ser-Gly-Val-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser-OH(SEQ ID NO: 85), as described above in Example 1.

[0276] The control monkeys had background titers ranging from 25 to 44,while the test group had titers of 1788 to 9588, as shown in Table 24below. TABLE 24 CONTROL GROUP TEST GROUP Monkey # Titer Monkey # Titer18782 30 18759 1788 18785 25 18789 5780 18786 30 18790 2718 18859 5418863 4139 18908 46 18945 9588 GMT 35 GMT 4068

[0277] C. Viral Challenge

[0278] At day 49 (week 7) after initial immunization, all monkeys weregiven 1 ml of a 1/1000 dilution of animal titered SHIV_(SF33) stockintravenously (challenge day 0). This corresponded to 50 animalinfectious doses_(50%) (50 AID₅₀) or 200 tissue culture infectiousdoses_(50%) (200 TCID₅₀).

[0279] D. Assessment of Infection

[0280] Plasma was drawn in EDTA at weeks 2, 4 and 8, and copies of viralRNA per ml of plasma were measured by QR-RT-PCR, using SIV probes forthe SIV component of SHIV_(SF33) (A. J. Conrad et al., J. Acq. Imm. Def.Syndrome and Hum. Retrovirol., 10:425 (1995)). The results aresummarized as follows in Tables 25 and 26. TABLE 25 CONTROLS SHIV RNAcopies/ml plasma Monkey # 2 weeks 4 weeks 8 weeks 18782 880,000 30,000<500 18785 610,000 80,000 <500 18786 500,000 50,000 <500 1885922,000,000 120,000 <500 18908 20,000,000 100,000 1,000 GMT 2,596,85167,869

[0281] TABLE 26 CONTROLS SHIV RNA copies/ml plasma Monkey # 2 weeks 4weeks 8 weeks 18759 920,000 60,000 <500 18789 950,000 50,000 <500 18790390,000 17,000 <500 18863 2,000,000 27,000 <500 18945 330,000 65,000 500GMT 742,034 38,938 INHIBITION: 71% 43% TEST VERSUS CONTROLS

[0282] As expected, SHIV_(SF33) caused an acute infection, with peaklevels of viral RNA at 2 weeks and barely or non-detectable levels byweek 8. Monkeys immunized with a synthetic peptide conjugate thatinduced antibodies to the Tat protein of the challenge SHIV_(SF33) virushad, by comparison with control immunized monkeys, a 71% reduction inpeak virus levels in plasma 2 weeks after viral challenge, with a 43%inhibition being still detectable in the subsiding plasma viral levelsat 4 weeks This shows that SHIV multiplication in vivo was inhibited inthe presence of antibodies to the Tat protein being utilized by thevirus, and suggests that a similar effect would prevail in HIV infectedhumans.

[0283] E. Assessment of Seroconversion

[0284] Subjects infected with HIV develop antibodies to virion surfaceproteins and this is detected by ELISA and used to diagnose infection.Monkey serums were tested prior to virus challenge and 8 weeks afterchallenge, using the HIVAB®HIV-1/HIV-2(rDNA)EIA (Abbott Labs, IL). Allpre-challenge serums were negative and all 8 week post challenge serumswere positive These findings provide additional support for the factthat antibodies to Tat protein do not register in diagnostic assays forHIV seroconversion.

EXAMPLE 10 Primate Animal Study

[0285] An additional monkey study was performed using immunization withsynthetic peptides coupled to diphtheria toxoid (9 monkeys) ordiphtheria toxoid immunized controls (5 monkeys). Initial immunizationwas with Freund's complete adjuvant, with boosts at 3, 6 and 9 weekswith incomplete Freund's adjuvant. The immunizing peptides wereCys-Val-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-His-Pro-Gly-Ser-amide (SEQ IDNO: 116) (Epitope I) andCys-Arg-Gln-Arg-Arg-Arg-Ala-Pro-Asp-Ser-Ser-Gln-Asn-His-Gln-OH (SEQ IDNO: 117) (Epitope III).

[0286] Monkeys were challenged with 50 AID₅₀ of SHIV33, as above, 2weeks after the last boost with immunogen. Geometric mean titers at 11weeks (time of challenge) in immunized animals were 46,000 for Epitope Iand 5,000 for Epitope III.

[0287] Geometric mean plasma viral loads at the peak (2 weeks) were456,000 for the test group and 234,000 for the controls. However, from 4weeks post challenge on viral loads in the test group droppedsignificantly below those in the control group (Table 27). TABLE 27 Week4 Week 8 Week 12 Week 16 GML Viral load - controls 17,000 900 400 120 (#negative) (0/5) (0/5) (0/5) (1/5) GML Viral load - tests 5,000 300 8 4(# negative) (0/9) (0/9) (6/9) (7/9) % inhibition 71% 67% 98% 97%

[0288] Seroconversion to SIV positive occured at 4-8 weeks. Thus thepost seroconversion viral loads (the most significant prognostic markerin HIV-1 infection) were significantly lowered in the presence ofantibodies to HIV-1 Tat protein.

[0289] In an effort to understand the high peak viral load at 2 weeks inthe test group, scrums were tested at the time of viral challenge (2weeks post the last immunization) for TNFα by ELISA. TNFα, which isreleased during an immune response, activates cells and is known toby-pass the requirement for Tat mediated activation to support HIV-1proliferation. The inventor determined that whereas serum TNFα wasundetectable pre-immunization, it was detected in all Tat immunizedmonkeys 2 weeks post immunization with a mean level of 7 pg/ml.

[0290] It was concluded that the effects of Tat interdiction on theacute infection were masked by the peri-immunization TNFα activation;once this subsided, the Tat immunized group developed significantlylower viral loads, with the majority having undetectable (<100copies/ml) levels in plasma.

EXAMPLE 11 Method and Kits for Detecting Titers and Specificities ofAntibodies Induced by Vaccination

[0291] To follow the titer and specificities of antibodies inducedfollowing immunization with the vaccines of this invention, an assaymethod may be employed. In one embodiment of such as assay, peptidescontaining the sequences reported in Table 28 (depending on thecomposition of the immunizing vaccine) are used to develop kitsmeasuring titers and reactivity patterns of antibodies in vaccinatedsubjects. TABLE 28 Epi- tope Sequence SEQ ID NOS I-Glu-Pro-Val-Asp-Pro-Arg- (amino acids 2-10 of Leu-Glu-Pro- SEQ IDNO: 1) I -Glu-Pro-Val-Asp-Pro-Lys- 118 Leu-Glu-Pro- I-Glu-Pro-Val-Asp-Pro-Ser- 119 Leu-Glu-Pro- I -Glu-Pro-Val-Asp-Pro-Asn-120 Leu-Glu-Pro- II -Lys-Gly-Leu-Gly-Ile-Ser- (amino acids 41-51 ofTyr-Gly-Arg-Lys-Lys- SEQ ID NO: 1) III -Arg-Arg-Ala-Pro-Pro-Asp-Ser-(amino acids 266-272 of SEQ ID NO: 3) III -Arg-Arg-Ala-His-Gln-Asp-Ser-121 III -Arg-Arg-Ala-Pro-Gln-Asp-Ser- 19 IV-Ser-Gln-Thr-His-Gln-Val-Ser- 122 Leu-Ser-Lys-Gln-Pro-

[0292] These peptides are synthesized with Biotin-Ser-Gly-Ser-Gly- (SEQID NO: 123) at the N-terminus. Each peptide is coated onto separateavidin coated plates, with a sequence -Ser-Gly-Ser-Gly- (SEQ ID NO: 30)serving as a spacer to ensure that the relevant peptide sequence isexternal to the biotin binding pocket of avidin. The plates are thenincubated with dilutions of test serum, washed, and the antibody bindingdetermined with reagent to human immunoglobulin, e.g., rabbit anti-humanimmunoglobulin, bound to, e.g., biotin, or directly labeled with enzyme.An avidin-enzyme complex is used to detect the biotin label, or areagent employed to react with the enzyme and produce a colorimetricsignal (R&D kit inserts).

[0293] Numerous modifications and variations of the present inventionare included in the above-identified specification and are expected tobe obvious to one of skill in the art. Such modifications andalterations to the compositions and processes of the present inventionare believed to be encompassed in the scope of the claims appendedhereto.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:124 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 72 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 1: Met Glu Pro Val Asp Pro Arg Leu Glu Pro TrpLys His Pro Gly Ser 1 5 10 15 Gln Pro Lys Thr Ala Cys Thr Asn Cys TyrCys Lys Lys Cys Cys Phe 20 25 30 His Cys Gln Val Cys Phe Thr Thr Lys GlyLeu Gly Ile Ser Tyr Gly 35 40 45 Arg Lys Lys Arg Arg Gln Arg Arg Arg AlaPro Gln Asp Ser Gln Thr 50 55 60 His Gln Val Ser Leu Ser Lys Gln 65 70(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 912 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A)NAME/KEY: CDS (B) LOCATION: join(1..876, 883..912) (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 2: GAG CTC TAC AAA TCC GGG GAT CCG GGT GAA GATCCG CGT TTA GAG CCG 48 Glu Leu Tyr Lys Ser Gly Asp Pro Gly Glu Asp ProArg Leu Glu Pro 1 5 10 15 TGG AAA CAC CCG GGT TCT GGT TCT GTT GAC CCTAAC CTT GAA CCT TGG 96 Trp Lys His Pro Gly Ser Gly Ser Val Asp Pro AsnLeu Glu Pro Trp 20 25 30 AAG CAT CCT GGC AGC TCC GGA GTC GAT CCC AAA CTCGAG CCC TGG AAA 144 Lys His Pro Gly Ser Ser Gly Val Asp Pro Lys Leu GluPro Trp Lys 35 40 45 CAC CCC GGA AGT TCG GGG GTA GAC CCA TCT CTG GAA CCATGG AAG CAT 192 His Pro Gly Ser Ser Gly Val Asp Pro Ser Leu Glu Pro TrpLys His 50 55 60 CCA GGG AGT GGT AGC GTG AAT CCG TCA TTA GAG CCG TGG AAACAC CCG 240 Pro Gly Ser Gly Ser Val Asn Pro Ser Leu Glu Pro Trp Lys HisPro 65 70 75 80 GGT TCA TCT GGA GTT GAT CCT CGC TTG GAA CCT TGG GAG CATCCT GGT 288 Gly Ser Ser Gly Val Asp Pro Arg Leu Glu Pro Trp Glu His ProGly 85 90 95 TCG TCC GGT GTA GAC CCC CGA CTT GAG CCC TGG AAT CAC CTC GGGAGT 336 Ser Ser Gly Val Asp Pro Arg Leu Glu Pro Trp Asn His Leu Gly Ser100 105 110 TCA GGC GTA GAT CAT CGG CTC GAA CCA TGG AAA CAT CCA GGT TCTGGA 384 Ser Gly Val Asp His Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gly115 120 125 GAT CTG CGC CAG CGG CGA CGT ACT CCT CAG GAT TCT GGA TCT CGACAA 432 Asp Leu Arg Gln Arg Arg Arg Thr Pro Gln Asp Ser Gly Ser Arg Gln130 135 140 CGT CGG CGC CCT CCC CAA GAC TCC TCA GGA CGG CAG CGC CGA CGACCC 480 Arg Arg Arg Pro Pro Gln Asp Ser Ser Gly Arg Gln Arg Arg Arg Pro145 150 155 160 CCA CAG GGT TCA GGT TCA CGT CAA CGA CGC GGT CCA CCC CAAGGC TCG 528 Pro Gln Gly Ser Gly Ser Arg Gln Arg Arg Gly Pro Pro Gln GlySer 165 170 175 GGT TCG CGC CAG CGG CGA CGT CCG CCT CAG AAC TCT AGT GGACGA CAA 576 Gly Ser Arg Gln Arg Arg Arg Pro Pro Gln Asn Ser Ser Gly ArgGln 180 185 190 CGT CGG CGC TCT CCC CAA GAT TCC GGC GGG CGG CAG CGC CGTCGA TCA 624 Arg Arg Arg Ser Pro Gln Asp Ser Gly Gly Arg Gln Arg Arg ArgSer 195 200 205 CCA CAG AAC TCA GGT GGG CGT CAA CGA CGC CGG ACT CCG CAATCT TCA 672 Pro Gln Asn Ser Gly Gly Arg Gln Arg Arg Arg Thr Pro Gln SerSer 210 215 220 TCC GGC CGC CAG CGG CGA CGT GCC CAT CAG AAT AGC GGC AGCCGA CAA 720 Ser Gly Arg Gln Arg Arg Arg Ala His Gln Asn Ser Gly Ser ArgGln 225 230 235 240 CGT CGG CGC GCA CAC CAA GAC AGC AGT GGG CGG CAG CGCCGT CGA GCG 768 Arg Arg Arg Ala His Gln Asp Ser Ser Gly Arg Gln Arg ArgArg Ala 245 250 255 CCT GAA GAT AGT GGT TCT CGT CAA CGA CGC CGG GCT CCCCCT GAC AGC 816 Pro Glu Asp Ser Gly Ser Arg Gln Arg Arg Arg Ala Pro ProAsp Ser 260 265 270 TCC GGA CGC CAG CGG CAA CGT GCA CCA GAT AGT TCC TCAGGT CAT CAC 864 Ser Gly Arg Gln Arg Gln Arg Ala Pro Asp Ser Ser Ser GlyHis His 275 280 285 CAC CAT CAT CAC TAATAA GAA TTC GGA TCC TCT AGA GTCGAC AAG CTT 912 His His His His Glu Phe Gly Ser Ser Arg Val Asp Lys Leu290 295 300 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 302 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 3: Glu Leu Tyr Lys Ser Gly Asp Pro Gly Glu Asp Pro Arg LeuGlu Pro 1 5 10 15 Trp Lys His Pro Gly Ser Gly Ser Val Asp Pro Asn LeuGlu Pro Trp 20 25 30 Lys His Pro Gly Ser Ser Gly Val Asp Pro Lys Leu GluPro Trp Lys 35 40 45 His Pro Gly Ser Ser Gly Val Asp Pro Ser Leu Glu ProTrp Lys His 50 55 60 Pro Gly Ser Gly Ser Val Asn Pro Ser Leu Glu Pro TrpLys His Pro 65 70 75 80 Gly Ser Ser Gly Val Asp Pro Arg Leu Glu Pro TrpGlu His Pro Gly 85 90 95 Ser Ser Gly Val Asp Pro Arg Leu Glu Pro Trp AsnHis Leu Gly Ser 100 105 110 Ser Gly Val Asp His Arg Leu Glu Pro Trp LysHis Pro Gly Ser Gly 115 120 125 Asp Leu Arg Gln Arg Arg Arg Thr Pro GlnAsp Ser Gly Ser Arg Gln 130 135 140 Arg Arg Arg Pro Pro Gln Asp Ser SerGly Arg Gln Arg Arg Arg Pro 145 150 155 160 Pro Gln Gly Ser Gly Ser ArgGln Arg Arg Gly Pro Pro Gln Gly Ser 165 170 175 Gly Ser Arg Gln Arg ArgArg Pro Pro Gln Asn Ser Ser Gly Arg Gln 180 185 190 Arg Arg Arg Ser ProGln Asp Ser Gly Gly Arg Gln Arg Arg Arg Ser 195 200 205 Pro Gln Asn SerGly Gly Arg Gln Arg Arg Arg Thr Pro Gln Ser Ser 210 215 220 Ser Gly ArgGln Arg Arg Arg Ala His Gln Asn Ser Gly Ser Arg Gln 225 230 235 240 ArgArg Arg Ala His Gln Asp Ser Ser Gly Arg Gln Arg Arg Arg Ala 245 250 255Pro Glu Asp Ser Gly Ser Arg Gln Arg Arg Arg Ala Pro Pro Asp Ser 260 265270 Ser Gly Arg Gln Arg Gln Arg Ala Pro Asp Ser Ser Ser Gly His His 275280 285 His His His His Glu Phe Gly Ser Ser Arg Val Asp Lys Leu 290 295300 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 4: Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 (2)INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 5: Gly Arg Gly Asp Ser Pro 1 5 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Val AspPro Arg Leu Glu Pro 1 5 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Val Asp Pro Lys Leu Glu Pro 1 5(2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 8: Val Asp Pro Ser Leu Glu Pro 1 5 (2)INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 9: Val Asp Pro Asn Leu Glu Pro 1 5 (2) INFORMATION FOR SEQ IDNO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Binding-site (B)LOCATION: 6 (D) OTHER INFORMATION: /note= “an amide is attached to theSer in position 6” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Trp Lys HisPro Gly Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Gly Pro Arg Leu Glu Pro 1 5(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 12: Ala Pro Arg Leu Glu Pro 1 5 (2) INFORMATIONFOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: His Pro Arg Leu Glu Pro 1 5 (2) INFORMATION FOR SEQ ID NO: 14: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid(C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Asp Pro Gly Leu GluPro 1 5 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:<Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 15: Asp Pro Arg Ile Glu Pro 1 5 (2) INFORMATIONFOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Asp Pro Arg Leu Gly Pro 1 5 (2) INFORMATION FOR SEQ ID NO: 17: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid(C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Asp Pro Arg Leu GluAla 1 5 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:<Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 18: Asn Pro Ser Leu Glu Pro 1 5 (2) INFORMATIONFOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Arg Arg Ala Pro Gln Asp Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 20:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: Gln Arg Arg ArgAla Pro Gln Asp Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: Gln Arg Arg Arg Ala His GlnAsp Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: Gln Arg Arg Arg Ala Pro ProAsp Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: Arg Arg Pro Pro Gln Asp Asn 15 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 24: Arg Arg Ala Pro Gln Asp Arg 1 5 (2)INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 25: Arg Gly Ala Pro Gln Asp Ser 1 5 (2) INFORMATION FOR SEQID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: Arg ArgAla Pro Glu Asp Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: Arg Arg Ala Ser Gln Asp Ser 15 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 13 (D) OTHER INFORMATION: /note=“Ser in position 13 is optionally modified with NH2” (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 28: Val Asp Pro Arg Leu Glu Pro Trp Lys His ProGly Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: Arg Gln Arg Arg Arg Ala ProGln Asp Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: Ser Gly Ser Gly 1 (2)INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 31: Val Asp Pro Arg Leu Pro Trp Lys His Pro Gly Ser 1 5 10(2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 32: Asp Pro Arg Leu Pro Trp Lys His Pro Gly Ser1 5 10 (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:<Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 33: Val Asp Pro Arg Leu Pro Trp 1 5 (2)INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 34: Val Asp Pro Arg Leu Pro 1 5 (2) INFORMATION FOR SEQ IDNO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: Val AspPro Arg Leu 1 5 (2) INFORMATION FOR SEQ ID NO: /note= “Val in position 2is optionally modified with a lower alkyl or a lower alkanoyl when Xaain the first position is absent.” (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 1 (D) OTHER INFORMATION: /note=“Xaa in the first position may be absent, or may be 1-5 aa, which may beoptionally modified with a lower alkyl or a lower alkanoyl, or Xaa maybe X-Pro, wherein X is Glu or Asp.” (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 2 (ix) FEATURE: (A) NAME/KEY: Modified-site(B) LOCATION: 8 (D) OTHER INFORMATION: /note= “Pro in position 8 isoptionally amidated, when it is the C terminal amino acid.” (ix)FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 9 (D) OTHERINFORMATION: /note= “The 9th position amino acid may be absent or asequence of 1-14 amino acids amidated at the carboxyl terminus.” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 36: Xaa Val Asp Pro Xaa Leu Glu Pro Xaa1 5 (2) INFORMATION FOR SEQ ID NO: /note= “Lys in position 12 isoptionally amidated, when it is the C terminal amino acid.” (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 1 (D) OTHERINFORMATION: /note= “Xaa in the first position may be absent or may beoptionally 1-5 amino acids, optionally modified with a lower alkyl orlower alkanoyl at the N terminus.” (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 2 (ix) FEATURE: (A) NAME/KEY: Modified-site(B) LOCATION: 12 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: Xaa Lys XaaLeu Gly Ile Ser Tyr Gly Arg Lys Lys Xaa 1 5 10 (2) INFORMATION FOR SEQID NO: /note= “Arg in position 2 is optionally modified with a loweralkyl or a lower alkanoyl, when it is the N terminal amino acid.” (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid(C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 1 (D)OTHER INFORMATION: /note= “The first position Xaa can be absent or asequence of 1-3 aa, optionally modified with a lower alkyl or loweralkanoyl; or it can be Gln-Arg, optionally modified with a lower alkylor lower alkanoyl.” (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 2 (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 5(D) OTHER INFORMATION: /note= “The 5th position amino acid can be Pro orHis.” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 6 (D)OTHER INFORMATION: /note= “The 6th position amino acid can be Gln orPro.” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 7 (D)OTHER INFORMATION: /note= “The 7th position amino acid can be Asp, Asn,Gly or Ser.” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 8(D) OTHER INFORMATION: /note= “Ser in position 8 is optionallyamidated.” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: Xaa Arg Arg Xaa XaaXaa Xaa Ser 1 5 (2) INFORMATION FOR SEQ ID NO: /note= “Ser in position 2is optionally modified with a lower alkyl or lower alkanoyl, when it isthe N terminal amino acid.” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 1 (D) OTHER INFORMATION: /note= “Xaa in thefirst position can be absent or is optionally 1-3 amino acids,optionally modified with a lower alkyl or lower alkanoyl.” (ix) FEATURE:(A) NAME/KEY: Modified-site (B) LOCATION: 2 (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 7 (D) OTHER INFORMATION: /note= “The 7thposition amino acid can be Ala or Val.” (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 13 (D) OTHER INFORMATION: /note= “Pro inposition 13 is optionally amidated when it is the C terminal aminoacid.” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 14 (D)OTHER INFORMATION: /note= “Xaa in position 14 can be absent or isoptionally 1-3 amino acids, optionally substituted with an amide.” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 39: Xaa Ser Gln Xaa His Gln Xaa Ser LeuSer Lys Gln Pro Xaa 1 5 10 (2) INFORMATION FOR SEQ ID NO: 40: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: Ser Gln Thr HisGln Ala Ser Leu Ser Lys Gln Pro 1 5 10 (2) INFORMATION FOR SEQ ID NO:41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: Asp HisArg Leu Glu Pro 1 5 (2) INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: Asp Pro Asn Leu Asp Pro 1 5(2) INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 43: Asp Pro Asn Ile Glu Pro 1 5 (2) INFORMATIONFOR SEQ ID NO: 44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: Asp Pro Asn Leu Glu Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 45: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45: Val Asp Pro LysLeu Glu Pro Trp Lys His Pro Gly Ser 1 5 10 (2) INFORMATION FOR SEQ IDNO: 46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: Val AspPro Ser Leu Glu Pro Trp Lys His Pro Gly Ser 1 5 10 (2) INFORMATION FORSEQ ID NO: 47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:Val Asp Pro Asn Leu Glu Pro Trp Lys His Pro Gly Ser 1 5 10 (2)INFORMATION FOR SEQ ID NO: 48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 48: Val Asn Pro Ser Leu Glu Pro Trp Lys His Pro Gly Ser 1 510 (2) INFORMATION FOR SEQ ID NO: 49: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 49: Val Asp His Arg Leu Glu Pro Trp Lys His ProGly Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 50: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: Arg Arg Ala Pro Pro Asp Asn 15 (2) INFORMATION FOR SEQ ID NO: 51: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 51: Arg Arg Ala Pro Gln Gly Asn 1 5 (2)INFORMATION FOR SEQ ID NO: 52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 52: Arg Gln Arg Arg Arg Ala His Gln Asn Ser 1 5 10 (2)INFORMATION FOR SEQ ID NO: 53: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 53: Arg Gln Arg Arg Arg Pro Pro Gln Asp Ser 1 5 10 (2)INFORMATION FOR SEQ ID NO: 54: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 54: Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His ProGly Ser 1 5 10 15 Gln Pro Lys Thr Ala 20 (2) INFORMATION FOR SEQ ID NO:55: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55: Phe IleThr Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg 1 5 10 15 GlnArg Arg Arg Ala Pro Gln Asp Ser Gln Thr His Gln Val Ser Leu 20 25 30 SerLys Gln Pro 35 (2) INFORMATION FOR SEQ ID NO: 56: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: Glu Pro Val Asp Pro Lys LeuGlu Pro 1 5 (2) INFORMATION FOR SEQ ID NO: 57: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: Val Asp Pro Lys Leu Gly Pro 15 (2) INFORMATION FOR SEQ ID NO: 58: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 4 (D) OTHER INFORMATION: /note=“Amino acid in position 4 can be Arg, Lys, Ser or Asn.” (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 58: Val Asp Pro Xaa Leu Glu Pro Tyr Lys His ProGly Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 59: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: Arg Gln Arg Arg Arg Pro ProGln Gly Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 60: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60: Arg Gln Arg Arg Gly Pro ProGln Gly Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 61: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: Arg Gln Arg Arg Arg Pro ProGln Asn Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 62: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: Arg Gln Arg Arg Arg Ser ProGln Asp Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 63: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: Arg Gln Arg Arg Arg Ser ProGln Asn Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 64: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: Arg Gln Arg Arg Arg Thr ProGln Ser Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 65: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65: Arg Gln Arg Arg Arg Ala HisGln Asp Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 66: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66: Arg Gln Arg Arg Arg Ala ProPro Asp Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 67: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 22 (D) OTHERINFORMATION: /note= “An amide is attached to the Lys in position 22.”(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: Cys Glu Pro Val Asp Pro LysLeu Glu Pro Trp Lys Glu Leu Gly Ile 1 5 10 15 Ser Tyr Gly Arg Lys Lys 20(2) INFORMATION FOR SEQ ID NO: 68: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 68: Arg Arg Thr Pro Gln Gly Ser 1 5 (2)INFORMATION FOR SEQ ID NO: 69: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 69: Arg Arg Ala Pro Gln Gly Ser 1 5 (2) INFORMATION FOR SEQID NO: 70: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70: Arg ArgThr Pro Gln Asp Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 71: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: Val Asp Pro Arg Leu Glu ProTrp Lys His Pro Gly Ser Gln Pro Lys 1 5 10 15 Thr Ala (2) INFORMATIONFOR SEQ ID NO: 72: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: Arg Arg Ala Pro Gln Asn Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 73:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: Arg Arg Ser ProGln Asp Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 74: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: Cys Arg Gln Arg Arg Arg AlaPro Gln Asp Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 75: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75: Arg Arg Pro Pro Gln Asp Ser 15 (2) INFORMATION FOR SEQ ID NO: 76: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 76: Arg Arg Pro Pro Gln Asn Ser 1 5 (2)INFORMATION FOR SEQ ID NO: 77: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 77: Arg Arg Pro Pro Gln Gly Ser 1 5 (2) INFORMATION FOR SEQID NO: 78: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78: Arg ArgSer Pro Gln Asn Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 79: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: Arg Arg Ala Pro Gln Ser Ser 15 (2) INFORMATION FOR SEQ ID NO: 80: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 80: Arg Arg Ser Pro Gln Gly Ser 1 5 (2)INFORMATION FOR SEQ ID NO: 81: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 81: Arg Arg Thr Pro Gln Asn Ser 1 5 (2) INFORMATION FOR SEQID NO: 82: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82: Arg GlnArg Gln Arg Ala Pro Asp Ser Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO:83: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83: Arg GlnArg Arg Arg Ala Pro Glu Asp Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO:84: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Binding-site (B)LOCATION: 13 (D) OTHER INFORMATION: /note= “an amide is attached to theSer in position 13” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84: Cys ValAsp Pro Asn Leu Glu Pro Trp His Pro Gly Ser 1 5 10 (2) INFORMATION FORSEQ ID NO: 85: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:Ser Gly Ser Gly Val Asp Pro Asn Leu Glu Pro Trp Lys His Pro Gly 1 5 1015 Ser (2) INFORMATION FOR SEQ ID NO: 86: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:<Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:(A) NAME/KEY: Modified-site (B) LOCATION: 4 (D) OTHER INFORMATION:/note= “Amino acid in position 4 can be Arg, Lys, Ser or Asn.” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 86: Val Asp Pro Xaa Leu Glu Pro 1 5 (2)INFORMATION FOR SEQ ID NO: /note= “Amino acid in position 2 can be Glyor Ala” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87: Lys Xaa Leu GlyIle Ser Tyr Gly Arg Lys Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 88:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 3(D) OTHER INFORMATION: /note= “Amino acid in position 3 can be Ala, Pro,Ser or Gln.” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 4(D) OTHER INFORMATION: /note= “Amino acid in position 4 can be Pro orHis.” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 5 (D)OTHER INFORMATION: /note= “Amino acid in position 5 can be Gln or Pro.”(ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 6 (D) OTHERINFORMATION: /note= “Amino acid in position 6 can be Asp, Asn, Gly orSer.” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88: Arg Arg Xaa Xaa Xaa XaaSer 1 5 (2) INFORMATION FOR SEQ ID NO: 89: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:<Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:(A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION:/note= “Amino acid in position 3 can be Asn or Thr.” (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 6 (D) OTHER INFORMATION: /note=“Amino acid in position 6 can be Ala or Val.” (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 89: Ser Gln Xaa His Gln Xaa Ser Leu Ser Lys Gln Pro 1 5 10(2) INFORMATION FOR SEQ ID NO: 90: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 90: Gly Ser Gly Ser 1 (2) INFORMATION FOR SEQ IDNO: 91: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 4 (D) OTHER INFORMATION: /note= “Biocytin-amide is attached toSer in position 4.” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91: Gly SerGly Ser 1 (2) INFORMATION FOR SEQ ID NO: /note= “NH2 is attached to theTrp in position 8” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 8 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:Val Asp Pro Arg Leu Glu Pro Trp 1 5 (2) INFORMATION FOR SEQ ID NO:/note= “NH2 is attached to Pro in position 7” (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 7 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 93: Val Asp Pro Arg Leu Glu Pro 1 5 (2)INFORMATION FOR SEQ ID NO: /note= “NH2 is attached to the Glu inposition 6” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 6 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94: Val Asp Pro ArgLeu Glu 1 5 (2) INFORMATION FOR SEQ ID NO: 95: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95: Val Asp Pro Arg Leu 1 5 (2)INFORMATION FOR SEQ ID NO: 96: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 96: Asp Pro Arg Leu Glu Pro Trp 1 5 (2) INFORMATION FOR SEQID NO: 97: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97: Gly ProArg Leu Glu Pro 1 5 (2) INFORMATION FOR SEQ ID NO: 98: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98: Ala Pro Arg Leu Glu Pro 1 5(2) INFORMATION FOR SEQ ID NO: 99: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 99: Glu Pro Val Asp Pro Arg Leu Glu Pro Trp LysHis Pro Gly Ser 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 100: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100: Val Asp Pro ArgLeu Glu Pro Trp Lys His Pro 1 5 10 (2) INFORMATION FOR SEQ ID NO: 101:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101: Val Asp Pro ArgLeu Glu Pro Trp Lys 1 5 (2) INFORMATION FOR SEQ ID NO: /note= “NH2 isattached to Pro in position 7” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 7 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102:Val Asp Pro Arg Leu Glu Pro 1 5 (2) INFORMATION FOR SEQ ID NO: 103: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103: Met Asp Pro ValAsp Pro Arg Leu Glu Pro 1 5 10 (2) INFORMATION FOR SEQ ID NO: 104: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104: Met Asp Pro ValAsp Pro Arg Leu Glu Pro Trp Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO:105: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105: PheIle Thr Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg 1 5 10 15Gln Arg (2) INFORMATION FOR SEQ ID NO: 106: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106: Lys Ala Leu Gly Ile Ser TyrGly Arg Lys Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: /note= “NH2 isattached to Ser in position 10” (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 10 (xi) SEQUENCE DESCRIPTION: SEQID NO: 107: Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser 1 5 10 (2)INFORMATION FOR SEQ ID NO: /note= “NH2 is attached to Ser in position 8”(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 8(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108: Gly Arg Arg Ala Pro Gln AspSer 1 5 (2) INFORMATION FOR SEQ ID NO: /note= “NH2 is attached to Gln inposition 11” (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified-site(B) LOCATION: 11 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109: Arg Arg AlaPro Gln Asp Ser Gln Thr His Gln 1 5 10 (2) INFORMATION FOR SEQ ID NO:110: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 3 (D) OTHER INFORMATION: /note= “Amino acid in position 3 canbe Ala, Pro, Ser or Gln” (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 6 (D) OTHER INFORMATION: /note= “Amino acid in position 6 canbe Asp, Asn, Gly or Ser” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110: ArgArg Xaa Pro Gln Xaa Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 111: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111: Ser Gln Thr HisGln Val Ser Leu Ser Lys Gln Pro Cys 1 5 10 (2) INFORMATION FOR SEQ IDNO: 112: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112: SerGln Thr His Gln Val Ser Leu Ser Lys Gln Pro 1 5 10 (2) INFORMATION FORSEQ ID NO: 113: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113:Ser Gln Asn His Gln Val Ser Leu Ser Lys Gln Pro 1 5 10 (2) INFORMATIONFOR SEQ ID NO: 114: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114: Ser Gln Thr His Gln Ala Ser Leu Ser Lys Gln Pro 1 5 10 (2)INFORMATION FOR SEQ ID NO: 115: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 115: Val Asp Pro Asn Leu Glu Pro Trp Lys His ProGly Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 116: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 14 (D) OTHERINFORMATION: /note= “An amide is attached to Ser in position 14” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 116: Cys Val Asp Pro Asn Leu Glu ProTrp Lys His Pro Gly Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 117: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117: Cys Arg Gln ArgArg Arg Ala Pro Asp Ser Ser Gln Asn His Gln 1 5 10 15 (2) INFORMATIONFOR SEQ ID NO: 118: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118: Glu Pro Val Asp Pro Lys Leu Glu Pro 1 5 (2) INFORMATION FOR SEQ IDNO: 119: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119: GluPro Val Asp Pro Ser Leu Glu Pro 1 5 (2) INFORMATION FOR SEQ ID NO: 120:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120: Glu Pro Val AspPro Asn Leu Glu Pro 1 5 (2) INFORMATION FOR SEQ ID NO: 121: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121: Arg Arg Ala His Gln Asp Ser 15 (2) INFORMATION FOR SEQ ID NO: 122: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 122: Ser Gln Thr His Gln Val Ser Leu Ser Lys GlnPro 1 5 10 (2) INFORMATION FOR SEQ ID NO: 123: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 1 (D) OTHERINFORMATION: /note= “Biotin is attached to Ser in position 1” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 123: Ser Gly Ser Gly 1 (2) INFORMATIONFOR SEQ ID NO: 124: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 7 (D) OTHER INFORMATION: /note= “Amino acidin position 7 can be Arg, Lys Ser or Asn” (xi) SEQUENCE DESCRIPTION: SEQID NO: 124: Glu Pro Val Asp Pro Xaa Leu Glu Pro 1 5

What is claimed is:
 1. A composition comprising a nucleic acid sequenceencoding at least two variants of the amino acid sequenceR1-Val-Asp-Pro-Y-Leu-Glu-Pro-R2 SEQ ID NO: 36, wherein each variant hasa different amino acid at Y, said Y being selected from the groupconsisting of Arg, Lys, Ser and Asn, wherein R1 is a sequence of up to 5amino acids, wherein R2 is a sequence of up to 14 additional aminoacids, or a fragment thereof, wherein said amino acid sequences orfragments induce antibodies that bind to HIV-1 Tat proteins fromdifferent HIV-1 strains and subtypes.
 2. The composition according toclaim 1, wherein at least one said amino acid sequence isR1-Val-Asp-Pro-Arg-Leu-Glu-Pro-R2.
 3. The composition according to claim1, wherein at least one said amino acid sequence isR1-Val-Asp-Pro-Lys-Leu-Glu-Pro-R2.
 4. The composition according to claim1, wherein at least one said amino acid sequence isR1-Val-Asp-Pro-Ser-Leu-Glu-Pro-R2
 5. The composition according to claim1, wherein at least one said amino acid sequence isR1-Val-Asp-Pro-Asn-Leu-Glu-Pro-R2.
 6. The composition according to claim1, wherein R1 is -X-Pro-, wherein X is selected from the groupconsisting of Glu or Asp.
 7. The composition according to claim 1,wherein R2 is selected from the group consisting of: -Trp, -Trp-Lys;-Trp-Lys-His; -Trp-Lys-His-Pro; -Trp-Lys-His-Pro-Gly; and-Trp-Lys-His-Pro-Gly-Ser-.
 8. The composition according to claim 1,wherein said nucleic acid sequence encodes said selected amino acidvariant sequences in an open reading frame with, or fused to, at leastone third nucleic acid sequence.
 9. The composition according to claim8, wherein said third amino acid sequence is selected from the groupconsisting of a second copy of one of said at least two variant aminoacid sequences or fragments, a third immunogenic amino acid sequencehaving a different sequence than said two variant amino acid sequences,and a carrier protein.
 10. The composition according to claim 1, whereinsaid molecule further comprises at least one regulatory nucleic acidsequences operatively linked to said nucleic acid sequence, which directand control expression of at least one said amino acid sequence encodedthereby in a host cell.
 11. The composition according to claim 1,wherein said nucleic acid sequence encodes sequentially at least threesaid variant amino acid sequences or fragments thereof, each varianthaving a different amino acid at position Y.
 12. The compositionaccording to claim 1, wherein said nucleic acid sequence encodessequentially at least four said variant amino acid sequences orfragments thereof, each variant having a different amino acid atposition Y.
 13. The composition according to claim 1, wherein saidnucleic acid sequence encodes multiple copies of the same amino acidsequence.
 14. The composition according to claim 1, wherein said nucleicacid sequence further encodes a spacer sequence interposed between eachamino acid sequence.
 15. The composition according to claim 1, whereinsaid molecule is a plasmid.
 16. The composition according to claim 1,wherein said molecule is a recombinant virus non-pathogenic to humans.17. The composition according to claim 1, wherein said molecule istransfected in a commensal bacterium non-pathogenic to humans.
 18. Thecomposition according to claim 1, further comprising a pharmaceuticalcarrier and an optional adjuvant.
 19. The composition according to claim18, which is a DNA vaccine.
 20. The composition according to claim 8,wherein said third amino acid sequence is selected from at least one ofthe group consisting of: (a) the amino acid sequenceR3-Lys-X-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-R4 of SEQ ID NO:37, whereinsaid amino acid X is Gly or Ala; wherein R3 is an optional sequence ofbetween 1 to about 5 additional amino acids; and wherein R4 is anoptional sequence of between about 1 to about 5 additional amino acids;or a fragment thereof; (b) at least one amino acid sequence of SEQ IDNOS: 11-18; (c) an amino acid sequence of R5-Arg-Arg-X-Z-A-Y-Ser- SEQ IDNO: 38, wherein X is selected from the group consisting of Ala, Pro,Ser, and Gln; wherein Y is selected from the group consisting of Asp,Asn, Gly and Ser; wherein Z is selected from the group consisting of Proand His; wherein A is selected from the group consisting of Gln and Pro;and wherein R5 is an optional sequence of between 1 and 3 amino acids;and (d) an amino acid sequence ofR7-Ser-Gln-X-His-Gln-Y-Ser-Leu-Ser-Lys-Gln-Pro-R8 SEQ ID NO: 39, whereinX is selected from the group consisting of Asn and Thr; wherein Y isselected from the group consisting of Ala and Val; wherein R7 is anoptional sequence of between 1 to 3 amino acids; and wherein R8 is asequence of about 1 to 3 amino acids.
 22. The composition according toclaim 21, wherein said third amino acid sequence is selected from amongthe sequences of SEQ ID NOS: 27, 40, 50, and
 51. 23. A method ofinducing an immune response in a mammal comprising administering to saidmammal an effective amount of a composition of claim
 19. 24. Acomposition comprising a nucleic acid sequence encoding an amino acidsequence R3-Lys-X-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys-R4 of SEQ ID NO:37, wherein said amino acid X is either Gly or Ala, wherein R3 is asequence of between 1 to about 5 additional amino acids; and wherein R4is a sequence of between about 1 to about 5 additional amino acids; or afragment thereof, wherein said amino acid sequence or fragment inducesantibodies that bind to HIV-1 Tat proteins from different HIV-1 strainsand subtypes.
 25. The composition according to claim 24, wherein saidfragment is Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-, amino acids 3 to 10 of SEQID NO:
 37. 26. The composition according to claim 24, wherein said aminoacid sequence is Lys-Gly-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-, amino acids 1to 10 of SEQ ID NO:
 37. 27. The composition according to claim 24,wherein said amino acid sequence isLys-Ala-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-, amino acids 1 to 10 of SEQ IDNO:
 37. 28. The composition according to claim 24 wherein said nucleicacid sequence encodes said selected amino acid sequence in an openreading frame with, or fused to, at least one second nucleic acidsequence.
 29. The composition according to claim 24 wherein said secondamino acid sequence is selected from the group consisting of a secondcopy of said first amino acid sequence or fragment, a second immunogenicamino acid sequence having a different sequence than said first aminoacid sequence, and a carrier protein.
 30. The composition according toclaim 24, wherein said molecule further comprises at least oneregulatory nucleic acid sequences operatively linked to said nucleicacid sequence, which direct and control expression of at least one saidamino acid sequence encoded thereby in a host cell.
 31. The compositionaccording to claim 24, wherein said nucleic acid sequence encodessequentially at least two amino acid sequences or fragments thereof,wherein each said at least two amino acid sequences or fragments induceantibodies that bind to HIV-1 Tat proteins from different HIV-1 strainsand subtypes.
 32. The composition according to claim 24, wherein saidnucleic acid sequence encodes multiple copies of the same amino acidsequence.
 33. The composition according to claim 24, wherein saidnucleic acid sequence encodes multiple different said amino acidsequences.
 34. The composition according to claim 24, wherein saidnucleic acid sequence further encodes a spacer sequence interposedbetween each amino acid sequence.
 35. The composition according to claim24, wherein said molecule is a plasmid.
 36. The composition according toclaim 24, wherein said molecule is a recombinant virus non-pathogenic tohumans.
 37. The composition according to claim 24, wherein said moleculeis transfected in a commensal bacterium non-pathogenic to humans. 38.The composition according to claim 24, further comprising apharmaceutical carrier and an optional adjuvant.
 39. The compositionaccording to claim 38, which is a DNA vaccine.
 40. A method of inducingan immune response in a mammal comprising administering to said mammalan effective amount of a composition of claim 39.